U.S. patent application number 14/436667 was filed with the patent office on 2016-10-13 for methods of using a fixed dose of a clotting factor.
This patent application is currently assigned to BIOGEN IDEC MA INC.. The applicant listed for this patent is BIOGEN IDEC MA INC.. Invention is credited to Haiyan JIANG.
Application Number | 20160296607 14/436667 |
Document ID | / |
Family ID | 50488800 |
Filed Date | 2016-10-13 |
United States Patent
Application |
20160296607 |
Kind Code |
A1 |
JIANG; Haiyan |
October 13, 2016 |
METHODS OF USING A FIXED DOSE OF A CLOTTING FACTOR
Abstract
The present invention provides methods of administering a
clotting factor by a fixed dosing regimen; methods of reducing,
ameliorating, or preventing one or more symptoms of a bleeding
disease or disorder; and a kit comprising a dotting factor useful
for a fixed dosing regimen. While plasma-derived and recombinant
clotting factor products allow hemophilia patients to live longer
and healthier, hemophilia still remains one of the most costly and
complex conditions to manage.
Inventors: |
JIANG; Haiyan; (US) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOGEN IDEC MA INC. |
Cambridge |
|
MA |
|
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
50488800 |
Appl. No.: |
14/436667 |
Filed: |
October 18, 2013 |
PCT Filed: |
October 18, 2013 |
PCT NO: |
PCT/US2013/065772 |
371 Date: |
April 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61715746 |
Oct 18, 2012 |
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61759856 |
Feb 1, 2013 |
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61760000 |
Feb 1, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/644 20130101;
A61K 38/4846 20130101; A61K 38/36 20130101; C07K 2319/30 20130101;
C12Y 304/21022 20130101 |
International
Class: |
A61K 38/48 20060101
A61K038/48; C12N 9/64 20060101 C12N009/64 |
Claims
1. A method of providing a clotting factor, comprising
administering a fixed dose of a clotting factor to a subject in
need thereof.
2. The method of claim 1, wherein the subject is in need of:
reducing, ameliorating, or preventing one or more symptoms of a
bleeding disease or disorder.
3. The method of claim 1, wherein the clotting factor is a modified
clotting factor.
4-8. (canceled)
9. The method of claim 1, wherein the fixed dose is administered at
regular intervals of every day, every two days, every three days,
twice a week, every four days, every five days, every six days,
every week, every eight days, every nine days, every 10 days, every
11 days, every 12 days, every 13 days, every two weeks, every three
weeks, or every four weeks.
10-13. (canceled)
14. The method of claim 1, wherein the body weight effect on
clearance (.theta..sub.BW.sub._.sub.CL) of the clotting factor is
equal to or less than about 0.75, 0.74, 0.73, 0.72, 0.71, 0.70,
0.69, 0.68, about 0.65, about 0.60, about 0.59, about 0.58, about
0.57, about 0.56, about 0.55, about 0.54, about 0.53, about 0.52,
about 0.51, about 0.50, about 0.49, about 0.48, about 0.47, about
0.46, about 0.45, about 0.44, about 0.43, about 0.42, about 0.41,
about 0.40, about 0.35, about 0.30, about 0.25, about 0.20, about
0.15, about 0.10, about 0.05, or about 0.
15. The method of claim 1, wherein the body weight effect on the
central volume of distribution (.theta..sub.BW.sub._.sub.V1) of the
clotting factor is equal to or less than about 0.75, 0.74, 0.73,
0.72, 0.71, 0.70, 0.69, 0.68, about 0.65, about 0.60, about 0.59,
about 0.58, about 0.57, about 0.56, about 0.55, about 0.54, about
0.53, about 0.52, about 0.51, about 0.50, about 0.49, about 0.48,
about 0.47, about 0.46, about 0.45, about 0.44, about 0.43, about
0.42, about 0.41, about 0.40, about 0.35, about 0.30, about 0.25,
about 0.20, about 0.15, about 0.10, about 0.05, or about 0.
16. (canceled)
17. The method of claim 14, wherein .theta..sub.BW.sub._.sub.CL of
the clotting factor is equal to or less than about 0.500.
18. The method of claim 15, wherein .theta..sub.BW.sub._.sub.V1 of
the clotting factor is equal to or less than about 0.467.
19-25. (canceled)
26. The method of claim 1, wherein the clotting factor is a
long-acting FIX polypeptide.
27-41. (canceled)
42. The method of claim 1, wherein the fixed dose is standard
across all body weights.
43-46. (canceled)
47. The method of claim 1, wherein the fixed dose is stratified
into two or more fixed dose amounts based on specified weight
categories.
48-90. (canceled)
91. The method of claim 1, wherein the fixed dose is about 2000 IU,
about 2,500 IU, about 3,000 IU, about 3,500 IU, or about 4,000 IU,
about 5,000 IU, about 7500 IU, or about 10000 IU.
92. The method of claim 1, wherein the fixed dose is administered
weekly or 10 days.
93-111. (canceled)
112. The method of claim 1, wherein the fixed dose is administered
intravenously or subcutaneously.
113. The method of claim 1, wherein the entire fixed dose is
administered.
114. The method of claim 1, wherein the fixed dose is provided in a
single vial.
115. The method of claim 1, wherein the fixed dose is provided in
two or more vials, the total contents of which provide the fixed
dosage amount.
116-119. (canceled)
120. A pharmaceutical composition comprising a fixed dose of a
modified clotting factor and a pharmaceutically acceptable carrier
for use to reduce, ameliorate, or prevent one or more symptoms of a
bleeding disease or disorder in a subject in need thereof.
121. (canceled)
122. A kit comprising the pharmaceutical composition of claim 120
and instructions to administer the fixed dose of the modified
clotting factor to the subject.
123-128. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to the field of
therapeutics for hemostatic disorders.
[0003] 2. Background Art
[0004] While plasma-derived and recombinant clotting factor
products allow hemophilia patients to live longer and healthier,
hemophilia still remains one of the most costly and complex
conditions to manage. The cost of clotting factor products exceeds
$50,000 a year per patient. See Blankenship C. S., Biotechnol.
Healthc. 2008, 5(4): 37-40. According to the National Heart, Lung,
and Blood Institute, National Institute of Health (NIH),
approximately 18,000 people in the U.S. have hemophilia, and 400
babies are born with the disease each year. Morbidity &
Mortality: 2012 Chart Book on Cardiovascular, Lung and Blood
Disease, page 5, National Heart, Lung, and Blood Institute, NIH.
Due to its complexity, this chronic disease requires a special
therapeutic management process for doctors, pharmacies, and
patients. Clinicians often assess lifestyle, psychosocial
requirements, and the home environment when evaluating a patient's
or guardian's ability to provide adequate care.
[0005] In hemophilia, blood clotting is disturbed by a lack of
certain plasma blood clotting factors. Hemophilia A, the most
common form of hemophilia, is caused by Factor VIII deficiency.
Hemophilia B is caused by decreased synthesis of Factor IX protein
or synthesis of defective Factor IX having reduced activity.
Treating hemophilia involves replacing missing or defective
clotting factor with recombinant or plasma-derived FVIII or FIX.
For patients who have developed antibodies against recombinant or
plasma-derived FVIII or FIX, Factor VII can be used as a bypass
therapy. Commercially available clotting factors are usually
administered by peripheral intravenous injection. However, for
patients with small veins or children who require frequent
injections, clotting factors can be administered by a central
venous access device. See Blankenship C. S., Biotechnol. Healthc.
2008, 5(4): 37-40.
[0006] Many biologics including clotting factors are administered
based on patient body size. Body sized-based dosing is assumed to
minimize inter-patient variability in pharmacokinetics (PK).
Currently, three FIX products are approved by the Food and Drug
Administration (FDA). The first, BENEFIX.RTM., is a recombinant FIX
product marketed by Pfizer. The second and third products are
plasma-derived FIX products, ALPHANINE.RTM. marketed by Grifols and
MONONINE.RTM. marketed by CSL Behring. According to their labels,
these three products are dosed based on individual body weight. In
particular, BENEFIX.RTM. is supplied as a lyophilized powder in
five different dosages: 250 IU, 500 IU, 1000 IU, 2000 IU, and 3000
IU. MONONINE.RTM. is supplied as a single dose vial with Sterile
Water for Injection at 500 IU and 1000 IU. ALPHANINE is supplied in
lyophilized form as single doses at 500 IU, 1000 IU, and 1500 IU.
The FIX dose required for each patient is calculated based on the
formula:
Number of factor IX IU required (IU)=Body Weight(kg).times.Desired
Factor IX Increase (% or IU/dL).times.Reciprocal of Observed
Recovery (IU/kg per IU/dL) (A)
[0007] Several Factor VIII products are also commercially
available, which include recombinant FVIII products (ADVATE.RTM.
and RECOMBINATE.RTM. marketed by Baxter, KOGENATE.RTM. FS marketed
by Bayer, HELIXATE.RTM. FS marketed by CSL-Behring, and XYNTHA.RTM.
and REFACTO.RTM. marketed by PFIZER) and Plasma-derived FVIII
products (HEMOFIL-M.RTM. marketed by Baxter, MONARC-M.RTM. by
American Red Cross, and MONOCLATE-P.RTM. marketed by CSL Behring).
The required FVIII dose for each patient is calculated using the
following formula:
Number of factor FVIII IU required (IU)=Body
Weight(kg).times.Desired Factor FVIII Increase (IU/dL or % of
normal).times.0.5(IU/kg per IU/dL) (B)
[0008] A Factor VII product, NOVOSEVEN.RTM. marketed by Novo
Nordisk, is also commercially available. The dosages of
NOVOSEVEN.RTM. are also calculated based on body weight: 90 g/kg
bolus injection every two hours for Hemophilia A or B with
inhibitors, 15-30 g/kg every 4-6 hours for congenital FVII
deficiency, or 70-90 g/kg every 2-3 hours for acquired hemophilia.
See NOVOSEVEN.RTM. label, page 1, January 2010, version 3, Novo
Nordisk A/S.
[0009] However, administering clotting factors via body
weight-based dosing can be inconvenient and costly for patients.
The invention as described herein provides improved clotting
factor-dosing strategies.
BRIEF SUMMARY OF THE INVENTION
[0010] In certain embodiments, the present invention provides a
method of providing a clotting factor comprising administering a
fixed dose of a clotting factor to a subject in need thereof. In
certain embodiments, a method of reducing, ameliorating, or
preventing one or more symptoms of a bleeding disease or disorder
in a subject comprising administering a fixed dose of a clotting
factor to a subject in need thereof is provided. In some aspects,
the clotting factor is a modified clotting factor. In some
embodiments, the modified clotting factor comprises a clotting
factor and a heterologous moiety, e.g., a heterologous moiety which
increases in vivo half-life of the clotting factor. In some aspects
the heterologous moiety is a non-polypeptide moiety or a
polypeptide moiety. In certain aspects, the heterologous moiety
comprises albumin, albumin binding polypeptide, an FcRn binding
partner, PAS, the C-terminal peptide (CTP) of the .beta. subunit of
human chorionic gonadotropin, polyethylene glycol (PEG),
hydroxyethyl starch (HES), albumin-binding small molecules, or
combinations thereof. In certain aspects, the modified clotting
factor is a long-acting clotting factor.
[0011] In some embodiments, the fixed dose of a clotting factor is
administered at regular intervals of every day, every two days,
every three days, twice a week, every four days, every five days,
every six days, every week, every eight days, every nine days,
every 10 days, every 11 days, every 12 days, every 13 days, every
two weeks, every three weeks, or every four weeks. In certain
embodiments, the fixed dose is administered as needed to control
bleeding.
[0012] In some aspects, the clotting factor has a wide therapeutic
window. For example, the therapeutic window for the clotting factor
can be a maximum serum concentration (C.sub.max) of about 150% of
normal and a minimum serum concentration (C.sub.min) of about 1% of
normal.
[0013] In other aspects, the clotting factor has a narrow
therapeutic window.
[0014] In certain embodiments provided herein, the body weight
effect on clearance (.theta..sub.BW.sub._.sub.CL) of the clotting
factor is equal to or less than about 0.75, 0.74, 0.73, 0.72, 0.71,
0.70, 0.69, 0.68, about 0.65, about 0.60, about 0.59, about 0.58,
about 0.57, about 0.56, about 0.55, about 0.54, about 0.53, about
0.52, about 0.51, about 0.50, about 0.49, about 0.48, about 0.47,
about 0.46, about 0.45, about 0.44, about 0.43, about 0.42, about
0.41, about 0.40, about 0.35, about 0.30, about 0.25, about 0.20,
about 0.15, about 0.10, about 0.05, or about 0. Alternatively, or
in addition, the body weight effect on the central volume of
distribution (.theta..sub.BW.sub._.sub.V1) of the clotting factor
is equal to or less than about 0.75, 0.74, 0.73, 0.72, 0.71, 0.70,
0.69, 0.68, about 0.65, about 0.60, about 0.59, about 0.58, about
0.57, about 0.56, about 0.55, about 0.54, about 0.53, about 0.52,
about 0.51, about 0.50, about 0.49, about 0.48, about 0.47, about
0.46, about 0.45, about 0.44, about 0.43, about 0.42, about 0.41,
about 0.40, about 0.35, about 0.30, about 0.25, about 0.20, about
0.15, about 0.10, about 0.05, or about 0.
[0015] In specific embodiments, the .theta..sub.BW.sub._.sub.CL of
the clotting factor is equal to or less than about 0.500 and/or the
.theta..sub.BW.sub._.sub.V1 of the clotting factor is equal to or
less than about 0.467. For example, in some embodiments the
.theta..sub.BW.sub._.sub.CL of the clotting factor is about 0.500
and/or the .theta..sub.BW.sub._.sub.V1 of the clotting factor is
about 0.467.
[0016] In some embodiments of the method provided herein the body
weight of the subject does not produce pharmacodynamic variability
within subjects. In other aspects, administration of a fixed dose
of the clotting factor results in reduced variability of
pharmacokinetic parameters across all body weights as compared to
administration of a body weight-based dose of the clotting factor.
For example, in certain embodiments the pharmacokinetic parameter
is area under the curve (AUC) and variability in AUC for a fixed
dose of the clotting factor is less than .+-.50%, less than
.+-.45%, less than .+-.40%, less than .+-.35%, less than .+-.30%,
or less than .+-.25% across all body weights.
[0017] In certain aspects of the method provided herein the
clotting factor is a long-acting FIX polypeptide. The long-acting
FIX polypeptide can include a FIX polypeptide and an FcRn binding
partner, and the FcRn binding partner can include an Fc region. The
long-acting FIX polypeptide can further include a second FcRn
binding partner, which can include a second Fc region. In certain
aspects the FcRn binding partner and the second FcRn binding
partner are associated, e.g., by a covalent bond, e.g., by a
disulfide bond. In other aspects the second FcRn binding partner is
not linked to an amino acid sequence by a peptide bond. In certain
embodiments, the long-acting FIX polypeptide is FIX monomer dimer
hybrid.
[0018] According to the present disclosure, the fixed dose of a
long acting FIX polypeptide can be standard across all body
weights, e.g., about 4000 IU per dose which is, e.g., administered
weekly, or about 8000 IU which is, e.g., administered weekly. In
other embodiments, the fixed dose is administered every 10
days.
[0019] In certain aspects a fixed dose of a long acting FIX
polypeptide is stratified into multiple (e.g., two or more) fixed
dose amounts based on specified weight categories, such as low body
weight, normal body weight, and high body weight. For example, the
fixed dose can be stratified into three fixed dose amounts suitable
for subjects with low, normal, or high body weight. In one
embodiment, the normal, low, or high body weight is determined
based on age, height, gender, frame size, general health, or any
combination thereof. In another embodiment, the normal, low, or
high body weight is determined independently of age, height,
gender, frame size, general health, or any combination thereof. In
other embodiments, the normal body weight for a human subject is
between about 50.+-.10 kg and about 100.+-.10 kg. In some
embodiments, the low body weight for a human subject is less than
about 50.+-.10 kg. In still other embodiments, the high body weight
for a human subject is greater than about 100.+-.10 kg.
[0020] In some aspects, the fixed dose is administered weekly
(i.e., once a week). In other aspects, the fixed dose is
administered every 10 days. In one embodiment, the subject has a
low body weight and the fixed dose is about 5000 IU per dose
administered every 10 days or about 6000 IU per dose administered
every 10 days. In another embodiment, the subject has a normal body
weight and the fixed dose is about 7500 IU per dose administered
every 10 days or about 8000 IU per dose administered every 10 days.
In other embodiments, the subject has a high body weight and the
fixed dose is about 10000 IU per dose administered every 10 days or
about 12000 IU per dose administered every 10 days.
[0021] In further aspects, the clotting factor is a long-acting
FVIII polypeptide. For example, the long-acting FVIII polypeptide
comprises a FVIII polypeptide and an FcRn binding partner, e.g., an
Fc region. In some embodiments, the long-acting FVIII polypeptide
further comprises a second FcRn binding partner, e.g., a second Fc
region. In one example, the FcRn binding partner and the second
FcRn binding partner are associated, e.g., by a covalent bond,
e.g., a disulfide bond. In another example, the long-acting FVIII
polypeptide is FVIII monomer dimer hybrid. In other examples, the
FVIII polypeptide in the long-acting polypeptide is a full-length
FVIII or a B-domain deleted FVIII.
[0022] In one aspect, the fixed dose is standard across all body
weights. In one embodiment, the fixed dose is administered twice
weekly. In another embodiment, the fixed dose is administered
weekly. In other embodiments, the fixed dose is stratified into
multiple (e.g., two or more) dose amounts based on specified weight
categories, e.g., low body weight, normal body weight, and high
body weight. In other embodiments, the fixed dose is stratified
into three dose sizes suitable for subjects with low, normal, or
high body weight. In some embodiments, the normal, low, or high
body weight is determined based on age, height, gender, frame size,
general health, or any combination thereof. In other embodiments,
the low, normal, or high body weight is determined independently of
age, height, gender, frame size, general health, or any combination
thereof. In one aspect, the normal body weight for a human subject
is between about 50.+-.10 kg and about 100.+-.10 kg. In another
aspect, the low body weight for a human subject is less than about
50.+-.10 kg. In other aspects, the high body weight for a human
subject is greater than about 100.+-.10 kg. In one example, the
fixed dose for the long-acting FVIII polypeptide is administered
twice weekly at about 2000 IU, about 2,500 IU, about 3,000 IU,
about 3,500 IU, or about 4,000 IU. In another example, the fixed
dose is administered weekly.
[0023] In some aspects, the fixed dose of the clotting factor is to
prevent one or more bleeding episodes. In one embodiment, the fixed
dose of the clotting factor is for individualized interval
prophylaxis of a bleeding episode. In another embodiment, the fixed
dose of the clotting factor is for on-demand or episodic treatment
of a bleeding episode. In other embodiments, the fixed dose of the
clotting factor is for perioperative management of a bleeding
episode. In certain embodiments, the subject is in need of
controlling or preventing bleeding or bleeding episodes, for
example, in need of peri-operative management or in need of
management of bleeding associated with surgery or dental
extraction. In some embodiments, the subject will undergo, is
undergoing, or has undergone major surgery. In certain embodiments,
the subject is in need of prophylactic treatment or in need of
on-demand treatment.
[0024] In other aspects, the fixed dose is provided in a single
container (e.g., vial) or in two or more containers (e.g., vials),
the total contents of which provide the fixed dosage amount.
[0025] The invention also includes use of a fixed dosage of a
clotting factor for the manufacture of a medicament for reducing,
ameliorating, or preventing one or more symptoms of a bleeding
disease or disorder in a subject in need thereof. The medicament
can be administered according to the method described herein.
[0026] Further included is a fixed dosage of a modified clotting
factor for use in reducing, ameliorating, or preventing one or more
symptoms of a bleeding disease or disorder in a subject in need
thereof. The fixed dosage of the invention is suitable for
administration according to the method described herein.
[0027] The present invention also includes a pharmaceutical
composition comprising a fixed dose of a modified clotting factor
and a pharmaceutically acceptable carrier for use to reduce,
ameliorate, or prevent one or more symptoms of a bleeding disease
or disorder in a subject in need thereof. The pharmaceutical
composition is suitable for administration according to the method
described herein.
[0028] The present invention further includes a kit comprising the
pharmaceutical composition described herein and instructions to
administer the fixed dose of the clotting factor to the subject. In
one embodiment, the entire fixed dose is administered. In another
embodiment, the fixed dose is provided in a single container (e.g.,
vial). In other embodiments, the fixed dose is provided in two or
more containers (e.g., vials), the total contents of which provide
the fixed dosage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows a diagram of the three-compartment model for
predicting population PK for rFIXFc. CL, clearance; V, volume of
distribution; Q, inter-compartmental clearance. V1 shows volume of
distribution in central compartment; and V2 and V3 show volume of
distribution in peripheral compartments. Q2 is inter-compartmental
clearance between V1 and V2. Q3 is inter-compartmental clearance
between V1 and V3.
[0030] FIG. 2A shows clearance (CL) estimates of baseline (week 1)
and repeat PK (week 26) profiles. FIG. 2B shows Volume of
Distribution of central compartment (V1) estimates of baseline
(week 1) and repeat PK (week 26) profiles. The thick line in the
middle of FIGS. 2A and 2B indicates mean, which did not change much
between two occasions.
[0031] FIGS. 3A to 3E show individual PK parameters versus body
weight (BW). FIG. 3A shows clearance in dL/h. FIG. 3B shows Volume
of Distribution of central compartment (V1) in dL. FIG. 3C shows
inter-compartmental clearance (Q2) in dL/h.
[0032] FIG. 3D shows Volume of Distribution in a peripheral
compartment (V2) in dL/h. FIG. 3E shows Volume of Distribution of a
peripheral compartment (V3).
[0033] FIG. 4A shows goodness-of-fit plots of FIX activity
predicted by the population PK model compared to observed FIX
activity. FIG. 4B shows goodness-of-fit plots of FIX activity
predicted by the individual PK model compared to observed FIX
activity.
[0034] FIG. 5A shows Visual Predictive Check (VPC) plots of the
population PK model for 50 IU/kg dose. FIG. 5B shows VPC plots of
the population PK model for 100 IU/kg dose. Gray and black lines
represent 10th, 50th, and 90th percentile of the simulated (gray)
and observed (black) data, respectively.
[0035] FIG. 6 shows validation of the population PK model with the
trough/peak records. R2=0.9857, P<0.001.
[0036] FIG. 7A shows the 97.5.sup.th, median, and 2.5.sup.th
percentiles of the simulated FIX activity-time profiles at steady
state in 1000 subjects following fixed dosing (4000 IU once weekly;
dotted line) compared with the 97.5.sup.th, median, and 2.5.sup.th
percentiles of the simulated FIX activity-time profiles at steady
state in 1000 subjects following BW-based dosing (50 IU/kg once
weekly; solid line). FIG. 7B shows the 97.5.sup.th median, and
2.5.sup.th percentiles of the simulated FIX activity-time profiles
at steady state in 1000 subjects following fixed dosing (8000 IU
every 10 days) compared with the 97.5.sup.th, median, and
2.5.sup.th percentiles of the simulated FIX activity-time profiles
at steady state in 1000 subjects following BW-based dosing (100
IU/kg every 10 days; solid lines).
[0037] FIG. 8 shows the percentiles of population within the target
therapeutic range following the fixed dosing and BW-based dosing
approaches in the BW-stratified populations.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention is derived from the recognition that a
fixed dosing regimen can be suitable for a clotting factor. The
present invention thus provides a method of administering a fixed
dose of a clotting factor to a subject in need thereof or a
population of two or more subjects in need thereof. Administration
of the fixed dose of the clotting factor can reduce, ameliorate, or
prevent one or more symptoms of a bleeding disease or disorder. For
example, administration of the fixed dose of the clotting factor
can control or prevent a bleeding episode. The invention also
includes a kit comprising one or more pharmaceutical compositions
and an instruction manual, wherein the one or more pharmaceutical
composition comprises a fixed dose of a clotting factor.
I. DEFINITIONS
[0039] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the context clearly dictates otherwise. The
terms "a" (or "an"), as well as the terms "one or more," and "at
least one" can be used interchangeably herein.
[0040] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term "and/or" as
used in a phrase such as "A and/or B" herein is intended to include
"A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the
term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following embodiments: A, B, and
C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B (alone); and C (alone).
[0041] The term "about" is used herein to mean approximately,
roughly, around, or in the regions of. When the term "about" is
used in conjunction with a numerical range, it modifies that range
by extending the boundaries above and below the numerical values
set forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
10 percent, up or down (higher or lower).
[0042] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0043] The term "polypeptide," "peptide" and "protein" are used
interchangeably and refer to a polymeric compound comprised of
covalently linked amino acid residues.
[0044] The term "polynucleotide" and "nucleic acid" are used
interchangeably and refer to a polymeric compound comprised of
covalently linked nucleotide residues. Polynucleotides may be DNA,
cDNA, RNA, single stranded, or double stranded, vectors, plasmids,
phage, or viruses. Polynucleotides include, but are not limited to,
those in Tables 4 and 6, which encode the polypeptides of Table 5
and 7. Polynucleotides also include fragments, variants, analogues,
or derivatives of the polynucleotides of Tables 4 and 6, e.g.,
those that encode fragments of the polypeptides of Table 5, 7, or
8.
[0045] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0046] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, amino acid sequences are written left to right in amino
to carboxy orientation. The headings provided herein are not
limitations of the various aspects or embodiments of the
disclosure, which can be had by reference to the specification as a
whole. Accordingly, the terms defined immediately below are more
fully defined by reference to the specification in its entirety.
Amino acids are referred to herein by either their commonly known
three letter symbols or by the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, are referred to by their commonly accepted single-letter
codes.
[0047] An "isolated" polypeptide, antibody, polynucleotide, vector,
cell, or composition refers to a polypeptide, antibody,
polynucleotide, vector, cell, or composition that is in a form not
found in nature. Isolated polypeptides, antibodies,
polynucleotides, vectors, cells or compositions include those which
have been purified to a degree that they are no longer in a form in
which they are found in nature. In some aspects, an antibody,
polynucleotide, vector, cell, or composition that is isolated is
substantially pure. In some aspects an antibody, polynucleotide,
vector, cell, or composition that is isolated is "recombinant."
[0048] The term "administering," as used herein, means to prescribe
or to give a pharmaceutically acceptable clotting factor to a
subject via a pharmaceutically acceptable route. Examples of routes
of administration include, but are not limited to, intravenous,
e.g., intravenous injection and intravenous infusion, e.g., via
central venous access. Additional routes of administration include
subcutaneous, intramuscular, oral, nasal, and pulmonary
administration. A clotting factor (e.g., a FIX or FVIII or modified
clotting factor protein) may be administered as part of a
pharmaceutical composition comprising at least one excipient.
[0049] The term "modified clotting factor" as used herein means a
clotting factor sequence that is modified in the polypeptide or
polynucleotide sequence by deletion, substitution, insertion,
conjugation, linkage, fusion, glycosylation, or any types of
modifications that are not present in the polypeptide sequences in
the wild-type clotting factor (e.g., FIX or FVIII) or the
commercially available clotting factor (e.g., REFACTO.RTM. or
XYNTHA.RTM. for SQ BDD FVIII; RECOMBINATE.RTM., ADVATE.RTM., OR
HELIXATE.RTM. for full-length FVIII; or BENEFIX.RTM.,
ALPHANINE.RTM., or MONONINE.RTM. for FIX).
[0050] The terms "long-acting" and "long-lasting" are used
interchangeably herein. In one embodiment, the term "long-acting"
or "long-lasting" indicates that the clotting activity as a result
of administration of a "long-acting" clotting factor is longer than
the clotting activity of a wild-type clotting factor (also referred
to as "short acting" or "shorter acting" clotting factor) (e.g.,
BENEFIX.RTM. or plasma-derived FIX ("pdFIX") for FIX, or SQ B
domain deleted FVIII (e.g., REFACTO.RTM.) or mature full-length
FVIII, e.g., RECOMBINATE.RTM., for FVIII). The "longer" clotting
activity can be measured by any known methods in the art, e.g.,
aPTT assay, chromogenic assay, ROTEM, TGA, and etc. In one
embodiment, the "longer" clotting activity can be shown by the
T.sub.1/2beta (activity). In another embodiment, the "longer"
clotting activity can be shown the level of the clotting factor
present in plasma, e.g., by the T.sub.1/2beta (antigen). In other
embodiments, the long-acting or long-lasting clotting factor works
longer in a coagulation cascade, e.g., is active for a longer
period, compared to a wild-type clotting factor (e.g., BENEFIX.RTM.
or plasma-derived FIX ("pdFIX") for FIX or SQ B domain deleted
FVIII (e.g., REFACTO.RTM.) or mature full-length FVIII, e.g.,
RECOMBINATE.RTM., for FVIII). The long-acting or long-lasting
clotting factor can comprise one or more heterologous moieties that
extend in vivo half-life of the clotting factor. Examples of the
heterologous moieties are described below.
[0051] The term "chimeric clotting factor" as used herein, means a
polypeptide that includes within it at least two polypeptides (or
portions thereof such as subsequences or peptides) from different
sources. Chimeric clotting factor can include two, three, four,
five, six, seven, or more polypeptides or portions thereof from
different sources, such as different genes, different cDNAs, or
different animal or other species. Chimeric clotting factors can
include one or more linkers joining the different polypeptides or
portions thereof. Thus, the polypeptides or portions thereof can be
joined directly or they may be joined indirectly, via linkers, or
both, within a single chimeric polypeptide. In certain embodiments,
chimeric clotting factors can include additional peptides such as
signal sequences and sequences such as 6His and FLAG that aid in
protein purification or detection. In addition, chimeric clotting
factors can have amino acid or peptide additions to the N- and/or
C-termini. Exemplary chimeric clotting factors of the invention are
Factor IX-Fc chimeric polypeptides or FVIII-Fc chimeric
polypeptides.
[0052] "Dosing interval," as used herein, means the amount of time
that elapses between multiple doses being administered to a
subject. Dosing interval can thus be indicated as a range. The
dosing interval in the methods of the invention using a clotting
factor can depend on the specific clotting factor. For example, a
dosing interval of a long-acting clotting factor can be at least
about one and a quarter, at least one and one-half to ten times
longer than the dosing interval required for an equivalent amount
(in IU/kg) of the wild-type clotting factor (i.e., a short-acting
clotting factor). The dosing interval when administering, e.g., a
Factor IX-Fc chimeric polypeptide (or a hybrid) of the invention
can be at least about three times longer than the dosing interval
required for an equivalent amount of said Factor IX without the
FcRn BP (defined below), e.g., Fc, portion (i.e., a polypeptide
consisting of said Factor IX). The dosing interval when
administering, e.g., a Factor VIII-Fc chimeric polypeptide (or a
hybrid) of the invention can be at least about one and a quarter,
at least one and one-half times longer than the dosing interval
required for an equivalent amount of the FVIII without the FcRn BP,
e.g., Fc, portion (i.e., a polypeptide consisting of the FVIII).
The dosing interval may be at least about one and one-half to
fifteen times longer than the dosing interval required for an
equivalent amount of the FIX or FVIII without, e.g., the Fc portion
(or a polypeptide consisting of the FIX or FVIII portion).
[0053] The term "dosing frequency" as used herein refers to the
frequency of administering doses of a clotting factor in a given
time. Dosing frequency can be indicated as the number of doses per
a given time, e.g., once a week or once in two weeks.
[0054] "Therapeutic dose," "dose," "effective dose," or "dosing
amount" as used herein, means a dose that achieves a plasma trough
level of a clotting activity at least about 1 IU/dl or above in the
subject administered with the clotting factor. For the purpose of
this invention, a "dose" can refer to the amount of the clotting
factor required to maintain a plasma trough level of a clotting
activity of at least about 1 IU/dl or above 1 IU/dl, at least about
2 IU/dl or above 2 IU/dl, at least about 3 IU/dl or above 3 IU/dl,
at least about 4 IU/dl or above 4 IU/dl, at least about 5 IU/dl or
above 5 IU/dl, at least about 6 IU/dl or above 6 IU/dl, at least
about 7 IU/dl or above 7 IU/dl, at least about 8 IU/dl or above 8
IU/dl, at least about 9 IU/dl or above 9 IU/dl, at least about 10
IU/dl or above 10 IU/dl, at least about 11 IU/dl or above 11 IU/dl,
at least about 12 IU/dl or above 12 IU/dl, at least about 13 IU/dl
or above 13 IU/dl, at least about 14 IU/dl or above 14 IU/dl, at
least about 15 IU/dl or above 15 IU/dl, or at least about 20 IU/dl
or above 20 IU/dl, throughout the administration of the clotting
factor. In another embodiment, the "dose" reduces or decreases the
frequency of bleeding or symptoms of a bleeding disorder. In other
embodiments, the "dose" stops on-going, uncontrollable bleeding or
bleeding episodes. In still other embodiments, the "dose" prevents
spontaneous bleeding or bleeding episodes in a subject susceptible
to such spontaneous bleeding or bleeding episodes. The "dose" or
"therapeutic dose" need not cure hemophilia.
[0055] The term "fixed dosing" or "fixed dose" as used herein means
a dosing amount given to a subject regardless of the body weight,
or who have a body weight within a given range. In one example, a
fixed dose can be given to any subjects in need thereof whether
they have a low body weight (e.g., lower than 10.sup.th percentile
of a body distribution), a normal body weight (e.g., between
10.sup.th percentile and 90.sup.th percentile of a body weight
distribution), or a high body weight (e.g., higher than 90.sup.th
percentile of a body weight distribution). In another example,
fixed dosing can be stratified over two or more patient
populations. For example, a first fixed dose can be given to a
subject having a low extreme body weight (e.g., lower than
10.sup.th percentile of a body weight distribution); a second fixed
dose can be given to a subject having a normal or high extreme body
weight (e.g., equal to or higher than 10.sup.th percentile of a
body weight distribution). In another example, fixed dosing can be
stratified over three or more groups, for example a first fixed
dose can be given to subjects having a low body weight (e.g., lower
than 10.sup.th percentile of a body weight distribution); a second
fixed dose can be given to subjects having a normal or high body
weight (e.g., equal to or higher than 10.sup.th percentile of a
body weight distribution), and a third fixed dose can be given to
subjects having a high body weight (e.g., higher than 90.sup.th
percentile of a body weight distribution).
[0056] The fixed dosing regimen can be stratified into two or more
fixed dose amounts based on specified weight categories. In one
embodiment, the weight categories are low body weight, normal body
weight, and high body weight. For example, the fixed dose can be
stratified into multiple fixed dose amounts (e.g., three) suitable
for subjects who fall within the weight categories, e.g., those
with low, normal, or high body weight. The ranges of each body
weight can be determined based on the patient's age, gender, frame
size, height, general health, or any combinations thereof or
independently of age, height, gender, frame size, general health,
or any combination there. A person of ordinary skill in the art can
assess the factors related to body weight and can determine the
specific body weight category for a subject.
[0057] The phrase "normal body weight" as used herein means a body
weight of a typical individual. Therefore, the phrase "normal body
weight" is used interchangeably with the phase "typical body
weight." In one example, a subject having a normal body weight is
neither obese nor underweight. In another example, a subject having
a normal body weight has a body weight between about 50 kg.+-.10 kg
and about 110 kg.+-.10 kg. In a particular example, a subject
having a normal body weight has a body weight between 57 kg and 104
kg. The normal body weight is above a low body weight and below a
high body weight.
[0058] The phrase "low body weight" as used herein means a body
weight that is lower than the body weight of a typical individual.
In one example, a subject having a low body weight is underweight.
In another example, a subject having a low body weight has a body
weight lower than about 50 kg.+-.10 kg. In other embodiments, a low
body weight is a low extreme body weight. In a particular example,
a subject having a low body weight has a body weight lower than
about 57 kg.
[0059] The phrase "high body weight" as used herein means a body
weight that is higher than the body weight of typical individual.
In one example, a subject having a high body weight is obese. In
another example, a subject having a high body weight has a body
weight higher than about 110 kg.+-.10 kg. In other embodiments, a
high body weight is a high extreme body weight. In a particular
example, a subject having a high body weight has a body weight
higher than about 104 kg.
[0060] The term "prophylaxis of one or more bleeding episodes,"
"prevent one or more bleeding episodes" or "prophylactic treatment"
as used herein means administering a clotting factor in fixed doses
to a subject over a course of time to increase the level of
clotting activity in a subject's plasma. In one embodiment,
"prophylaxis of one or more bleeding episodes" or "prevent one or
more bleeding episodes" indicates use of a clotting factor to
prevent or inhibit occurrence of one or more spontaneous or
uncontrollable bleeding or bleeding episodes or to reduce the
frequency of one or more spontaneous or uncontrollable bleeding or
bleeding episodes. "Routine prophylaxis" is used to prevent or
reduce the frequency of bleeding episodes in subjects with
hemophilia A or B. In another embodiment, the increased clotting
activity level is sufficient to decrease the incidence of
spontaneous bleeding or to prevent bleeding in the event of an
unforeseen injury. Prophylactic treatment decreases or prevents
bleeding episodes, for example, those described under on-demand
treatment.
[0061] The term "about once a week" as used herein means
approximate number, and "about once a week" can include every seven
days.+-.two days, i.e., every five days to every nine days. The
dosing frequency of "once a week" thus can be every five days,
every six days, every seven days, every eight days, or every nine
days.
[0062] The term "individualized interval prophylaxis" as used
herein means use of a long-acting clotting factor for an
individualized dosing interval or frequency for a subject to
prevent or inhibit occurrence of one or more spontaneous and/or
uncontrollable bleeding or bleeding episodes or to reduce the
frequency of one or more spontaneous and/or uncontrollable bleeding
or bleeding episodes to the subject. In one embodiment, the
"individualized interval" includes every 10 days.+-.3 days, i.e.
every seven days to every 13 days. The dosing frequency of the
"individualized interval prophylaxis" thus can be every seven days,
every eight days, every nine days, every ten days, every 11 days,
every 12 days, or every 13 days.
[0063] The term "on-demand treatment," as used herein, means
treatment that is intended to take place over a short course of
time and is in response to an existing condition, such as a
bleeding episode, or a perceived short term need such as planned
surgery. The "on-demand treatment" is used interchangeably with
"episodic" treatment. Conditions that may require on-demand
treatment include a bleeding episode, hemarthrosis, muscle bleed,
oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,
trauma, trauma capitis, gastrointestinal bleeding, intracranial
hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage,
bone fracture, central nervous system bleeding, bleeding in the
retropharyngeal space, bleeding in the retroperitoneal space, or
bleeding in the illiopsoas sheath. Bleeding episodes other than
these known in the art are also included. The subject can be in
need of surgical prophylaxis, peri-operative management, or
treatment for surgery. Such surgeries include, but are not limited
to, minor surgery, major surgery, tooth extraction, tonsillectomy,
other dental/thoraco-facial surgeries, inguinal herniotomy,
synovectomy, total knee replacement, other joint replacement,
craniotomy, osteosynthesis, trauma surgery, intracranial surgery,
intra-abdominal surgery, intrathoracic surgery. Surgeries other
than these are also included.
[0064] Additional non-limiting conditions that can require
on-demand treatment include minor hemorrhage, hemarthroses,
superficial muscle hemorrhage, soft tissue hemorrhage, moderate
hemorrhage, intramuscle or soft tissue hemorrhage with dissection,
mucous membrane hemorrhage, hematuria, major hemorrhage, hemorrhage
of the pharynx, hemorrhage of the retropharynx, hemorrhage of the
retroperitonium, hemorrhage of the central nervous system, bruises,
cuts, scrapes, joint hemorrhage, nose bleed, mouth bleed, gum
bleed, intracranial bleeding, intraperitoneal bleeding, minor
spontaneous hemorrhage, bleeding after major trauma, moderate skin
bruising, or spontaneous hemorrhage into joints, muscles, internal
organs or the brain. Additional reasons for on-demand treatment
include the need for peri-operative management for surgery or
dental extraction, major surgery, extensive oral surgery, urologic
surgery, hernia surgery, orthopedic surgery such as replacement of
knee, hip, or other major joint.
[0065] The term "treatment" or "treating" as used herein means
amelioration or reduction of one or more symptoms of bleeding
diseases or disorders including, but not limited to, hemophilia A
or hemophilia B. In one embodiment, "treatment of" or "treating" a
bleeding disease or disorder includes prevention of one or more
symptoms of a bleeding disease or disorder. In a bleeding disease
or disorder caused by a clotting factor deficiency (e.g., a low
baseline clotting activity), the term "treatment" or "treating"
means a clotting factor replacement therapy. By administering a
clotting factor to a subject, the subject can achieve and/or
maintain a plasma trough level of a clotting activity at about 1
IU/dl or above 1 IU/dl. In other embodiments, "treatment" or
"treating" means reduction of the frequency of one or more symptoms
of bleeding diseases or disorders, e.g., spontaneous or
uncontrollable bleeding episodes. "Treatment," however, need not be
a cure.
[0066] The term "perioperative management" as used herein means use
of a clotting factor before, concurrently with, or after an
operative procedure, e.g., a surgical operation. The use for
"perioperative management" of one or more bleeding episode includes
surgical prophylaxis before (i.e., preoperative), during (i.e.,
intraoperative), or after (i.e., postoperative) a surgery to
prevent one or more bleeding or bleeding episode or reducing or
inhibiting spontaneous and/or uncontrollable bleeding episodes
before, during, and after a surgery.
[0067] Pharmacokinetic (PK) parameters include the terms above and
the following terms, which have their ordinary meaning in the art,
unless otherwise indicated. Some of the terms are explained in more
detail in the Examples. PK parameters can be based on clotting
factor antigen level (often denoted parenthetically herein as
"antigen") or clotting activity level (often denoted
parenthetically herein as "activity"). In the literature, PK
parameters are often based on clotting activity level due to the
presence in the plasma of some patients of endogenous, inactive
clotting factor, which interferes with the ability to measure
administered (i.e., exogenous) clotting factor using antibody
against clotting factor. However, when a clotting factor is
administered, clotting factor antigen can be accurately measured
using antibody to the heterologous polypeptide. In addition,
certain PK parameters can be based on model predicted data (often
denoted parenthetically herein as "model predicted") or on observed
data (often denoted parenthetically herein as "observed").
[0068] "Baseline," as used herein, is the lowest measured plasma
clotting factor level in a subject prior to administering a dose.
The clotting factor plasma levels can be measured at two time
points prior to dosing: at a screening visit and immediately prior
to dosing. Alternatively, (a) the baseline in patients whose
pretreatment clotting activity is <1% of normal, who have no
detectable clotting factor antigen, and have nonsense genotypes can
be defined as 0%, (b) the baseline for patients with pretreatment
clotting activity <1% of normal and who have detectable clotting
factor antigen can be set at 0.5%, (c) the baseline for patients
whose pretreatment clotting activity is between 1-2% is C.sub.min
(the lowest activity throughout the PK study), and (d) the baseline
for patients whose pretreatment clotting activity is .gtoreq.2% can
be set at 2% of normal. Activity above the baseline pre-dosing can
be considered residue drug from prior treatment, and can be decayed
to baseline and subtracted from the PK data following clotting
factor dosing.
[0069] "Trough," as used herein, is the lowest plasma clotting
activity level reached after administering a dose of a clotting
factor molecule (e.g., chimeric clotting factor) and before the
next dose is administered, if any. Trough is used interchangeably
herein with "threshold." Baseline clotting factor levels are
subtracted from measured clotting factor levels to calculate the
trough level.
[0070] "Subject," as used herein means a mammal. The subject can be
a human, e.g., a human patient. Subject as used herein includes an
individual who is known to have at least one incidence of
uncontrolled bleeding episodes, who has been diagnosed with a
disease or disorder associated with uncontrolled bleeding episodes,
e.g., a bleeding disease or disorder, e.g., hemophilia A or
hemophilia B, who are susceptible to uncontrolled bleeding
episodes, e.g., hemophilia, or any combinations thereof. Subjects
can also include an individual who is in danger of one or more
uncontrollable bleeding episodes prior to a certain activity, e.g.,
a surgery, a sport activity, or any strenuous activities. The
subject can have a baseline clotting activity less than 1%, less
than 0.5%, less than 2%, less than 2.5%, less than 3%, or less than
4%.
[0071] "Variant," as used herein, refers to a polynucleotide or
polypeptide differing from the original polynucleotide or
polypeptide, but retaining essential properties thereof, e.g.,
clotting activity or Fc (FcRn binding) activity. Generally,
variants are overall closely similar, and, in many regions,
identical to the original polynucleotide or polypeptide. Variants
include polypeptide and polynucleotide fragments, deletions,
insertions, and modified versions of original polypeptides.
II. CLOTTING FACTORS
[0072] The present invention is directed to a clotting factor
suitable for a fixed dosing regimen. A suitable dosing strategy can
be identified for a particular drug based on its pharmacokinetic
(PK) and/or pharmacodynamic (PD) properties. For example, a good
drug candidate for a fixed dosing strategy provides more consistent
exposure of the drug across subjects when administered by a fixed
dosing regimen rather than by a body weight based dosing regimen.
Advantages of the present invention include: improved regimen
compliance; reduced break through bleeds; increased protection of
joints from bleeds; prevention of joint damage; reduced morbidity;
reduced mortality; prolonged protection from bleeding; decreased
thrombotic events; and improved quality of life. In one embodiment,
a clotting factor suitable for a fixed dosing regimen exhibits a
wide therapeutic window. In another embodiment, a clotting factor
suitable for a fixed dosing regimen has less inter-individual
variability of pharmacokinetic parameters (e.g., AUC or C.sub.max)
when administered by a fixed dosing regimen compared to the
inter-individual variability of pharmacokinetic parameters when
administered by a body-weight based dosing regimen. In another
embodiment, a clotting factor suitable for a fixed dosing regimen
has inter-individual variability of pharmacokinetic parameters
(e.g., AUC or C.sub.max) that is similar when administered either
by a fixed dosing regimen or by a body-weight based dosing
regimen.
[0073] In one aspect, the pharmaceutical properties of a clotting
factor suitable for a fixed dosing regimen can be represented by
the following formulas:
AUC=Dose/CL, (C)
CL=Typical CL.times.(BW/Typical BW).sup.exponent (D)
Cmax=Dose/V, (E)
V=Typical V.times.(BW/TypicalBW).sup.exponent (F)
[0074] The exponent for formula (D) indicates a body weight effect
on clearance (.theta..sub.BW.sub._.sub.CL). The exponent for
formula (F) indicates a body weight effect on the central volume of
distribution (.theta..sub.BW.sub._.sub.V1). In another aspect of
the invention, the body weight effect on clearance
(.theta..sub.BW.sub._.sub.CL) of the clotting factor is equal to or
less than 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67,
0.66, 0.65, 0.64, 0.63, 0.62, 0.61, 0.6, 0.59, 0.58, 0.57, 0.56,
0.55, 0.54, 0.53, 0.52, 0.51, 0.50, 0.49, 0.48, 0.47, 0.46, 0.45,
0.44, 0.43, 0.42, 0.41, 0.40, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34,
0.32, 0.31, 0.3, 0.25, 0.2, 0.15, 0.1, 0.5, or 0. In other aspects,
the body weight effect on the central volume of distribution
(.theta..sub.BW.sub._.sub.V1) of the clotting factor is equal to or
less than 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67,
0.66, 0.65, 0.64, 0.63, 0.62, 0.61, 0.6, 0.59, 0.58, 0.57, 0.56,
0.55, 0.54, 0.53, 0.52, 0.51, 0.50, 0.49, 0.48, 0.47, 0.46, 0.45,
0.44, 0.43, 0.42, 0.41, 0.40, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34,
0.32, 0.31, 0.3, 0.25, 0.2, 0.15, 0.1, 0.5, or 0. In still other
aspects, .theta..sub.BW.sub._.sub.CL is equal to or less than 0.75,
0.74, 0.73, 0.72, 0.71, 0.70, 0.69, 0.68, 0.67, 0.66, 0.65, 0.64,
0.63, 0.62, 0.61, 0.6, 0.59, 0.58, 0.57, 0.56, 0.55, 0.54, 0.53,
0.52, 0.51, 0.50, 0.49, 0.48, 0.47, 0.46, 0.45, 0.44, 0.43, 0.42,
0.41, 0.40, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.32, 0.31, or 0.3,
0.25, 0.2, 0.15, 0.1, 0.5, or 0 and .theta..sub.BW.sub._.sub.V1 is
equal to or less than 0.75, 0.74, 0.73, 0.72, 0.71, 0.70, 0.69,
0.68, 0.67, 0.66, 0.65, 0.64, 0.63, 0.62, 0.61, 0.6, 0.59, 0.58,
0.57, 0.56, 0.55, 0.54, 0.53, 0.52, 0.51, 0.50, 0.49, 0.48, 0.47,
0.46, 0.45, 0.44, 0.43, 0.42, 0.41, 0.40, 0.39, 0.38, 0.37, 0.36,
0.35, 0.34, 0.32, 0.31, or 0.3, 0.25, 0.2, 0.15, 0.1, 0.5, or 0. In
some aspects, the clotting factor has .theta..sub.BW.sub._.sub.CL
equal to or less than about 0.500 and .theta..sub.BW.sub._.sub.V1
equal to or less than 0.467. In other embodiments, the clotting
factor has .theta..sub.BW.sub._.sub.CL equal to about 0 and
.theta..sub.BW.sub._.sub.V1 equal to or less than 0.492. In other
embodiments, the clotting factor has .theta..sub.BW.sub._.sub.CL
equal to or less than about 0.436 and .theta..sub.BW.sub._.sub.V1
equal to or less than about 0.396.
[0075] In certain aspects, a clotting factor is administered to a
population of two or more subjects. In some aspects, the area under
curve (AUC) or C.sub.max between a high extreme body weight subject
and a low extreme body weight subject after administration of the
fixed dosing of the clotting factor is similar to or less variable
than AUC or C.sub.max between a high body weight subject and a low
body weight after administration of a body weight-based dosing
amount of the clotting factor. In one embodiment, the variability
in AUC is less than .+-.50%, less than .+-.45%, less than .+-.40%,
less than .+-.35%, less than .+-.30%, or less than .+-.25%. In
another embodiment, the variability in C.sub.max is less than
.+-.50%, less than .+-.45%, less than .+-.40%, less than .+-.35%,
less than .+-.30%, or less than .+-.25%.
[0076] In other aspects, the clotting factor has a wide therapeutic
window. In one embodiment, the therapeutic window for the clotting
factor comprises a maximum serum concentration (C.sub.max) of about
150% of normal and a minimum serum concentration (C.sub.min) of
about 1% of normal. In still other aspects, the body weight of the
subject does not drive pharmacodynamic variability when
administered by a fixed dosing regimen compared to the
pharmacodynamics variability when administered by a body weight
based dosing regimen. In some aspects, a clotting factor of the
invention has low or no off-target toxicity. In certain aspects, a
clotting factor is cleared primarily through cellular uptake in
liver.
[0077] In some aspects, a clotting factor has less pharmacokinetic
inter-subject variability than a clotting factor suitable for a
body-weight based dosing. In one embodiment, the inter-subject
variability is about 20% to about 50%, about 21% to about 49%,
about 22% to about 48%, about 23% to about 47%, about 24% to about
46%, about 25% to about 46%, about 26% to about 46% for total
clearance (CL) and Volume of Distribution at Steady State
(Vss).
[0078] A clotting factor suitable for a fixed dosing strategy can
be a wild-type clotting factor, a commercially available clotting
factor, or a modified clotting factor. Examples of the wild-type
clotting factors include, but are not limited to, full-length
recombinant FIX (e.g., BENEFIX.RTM.), plasma-derived FIX (e.g.,
ALPHANINE.RTM., or MONONINE.RTM.), or full-length recombinant FVIII
(e.g., RECOMBINATE.RTM., ADVATE.RTM., or HELIXATE.RTM.), or
B-domain deleted recombinant FVIII (e.g., REFACTO.RTM. or
XYNTHA.RTM.).
[0079] Clotting factors for the invention can be modified. Modified
clotting factors includes any clotting factors that have
improvements in one or more aspects, pharmacokinetics (PK),
pharmacodynamics (PD), stability, expression, or any combinations
thereof. In one embodiment, a modified clotting factor comprises a
clotting factor and a heterologous moiety. In another embodiment, a
clotting factor for the invention is a long-acting clotting factor.
Long-acting clotting factors can comprise a heterologous moiety
that increases in vivo half-life of the clotting factor. In other
embodiments, the heterologous moiety for the modified clotting
factor (e.g., long-acting clotting factor) is a polypeptide moiety
or a non-polypeptide moiety. In yet other embodiments, a
heterologous moiety comprises albumin, albumin binding polypeptide,
an FcRn binding partner, PAS, the C-terminal peptide (CTP) of the
.beta. subunit of human chorionic gonadotropin, polyethylene glycol
(PEG), hydroxyethyl starch (HES), albumin-binding small molecules,
or combinations thereof. Examples of the heterologous moieties are
described below. In some embodiments, a clotting factor of the
invention is a chimeric clotting factor. In some embodiments, the
heterologous moiety is linked to the N-terminus or the C-terminus
of the FVIII polypeptide or inserted between two amino acids of the
FVIII polypeptide.
[0080] A. Factor IX
[0081] In certain embodiments, a clotting factor of the invention
is a modified FIX polypeptide. In one example, a modified clotting
factor useful for the invention comprises a long-acting FIX
polypeptide, which is a chimeric polypeptide comprising a FIX
polypeptide and an FcRn binding partner. The FIX polypeptide of the
invention comprises a functional Factor IX polypeptide in its
normal role in coagulation, unless otherwise specified. Thus, the
FIX polypeptide includes variant polypeptides that are functional
and the polynucleotides that encode such functional variant
polypeptides. In one embodiment, the FIX polypeptides are the
human, bovine, porcine, canine, feline, and murine FIX
polypeptides. The full-length polypeptide and polynucleotide
sequences of FIX are known, as are many functional variants, e.g.,
fragments, mutants and modified versions. FIX polypeptides include
full-length FIX, full-length FIX minus Met at the N-terminus,
full-length FIX minus the signal sequence, mature FIX (minus the
signal sequence and propeptide), and mature FIX with an additional
Met at the N-terminus. FIX can be made by recombinant means
("recombinant Factor IX" or "rFIX"), i.e., it is not naturally
occurring or derived from plasma.
[0082] A great many functional FIX variants are known.
International publication number WO 02/040544 A3, which is herein
incorporated by reference in its entirety, discloses mutants that
exhibit increased resistance to inhibition by heparin at page 4,
lines 9-30 and page 15, lines 6-31. International publication
number WO 03/020764 A2, which is herein incorporated by reference
in its entirety, discloses FIX mutants with reduced T cell
immunogenicity in Tables 2 and 3 (on pages 14-24), and at page 12,
lines 1-27. International publication number WO 2007/149406 A2,
which is herein incorporated by reference in its entirety,
discloses functional mutant FIX molecules that exhibit increased
protein stability, increased in vivo and in vitro half-life, and
increased resistance to proteases at page 4, line 1 to page 19,
line 11. WO 2007/149406 A2 also discloses chimeric and other
variant FIX molecules at page 19, line 12 to page 20, line 9.
International publication number WO 08/118507 A2, which is herein
incorporated by reference in its entirety, discloses FIX mutants
that exhibit increased clotting activity at page 5, line 14 to page
6, line 5. International publication number WO 09/051717 A2, which
is herein incorporated by reference in its entirety, discloses FIX
mutants having an increased number of N-linked and/or O-linked
glycosylation sites, which results in an increased half-life and/or
recovery at page 9, line 11 to page 20, line 2. International
publication number WO 09/137254 A2, which is herein incorporated by
reference in its entirety, also discloses Factor IX mutants with
increased numbers of glycosylation sites at page 2, paragraph [006]
to page 5, paragraph [011] and page 16, paragraph [044] to page 24,
paragraph [057]. International publication number WO 09/130198 A2,
which is herein incorporated by reference in its entirety,
discloses functional mutant FIX molecules that have an increased
number of glycosylation sites, which result in an increased
half-life, at page 4, line 26 to page 12, line 6. International
publication number WO 09/140015 A2, which is herein incorporated by
reference in its entirety, discloses functional FIX mutants that an
increased number of Cys residues, which may be used for polymer
(e.g., PEG) conjugation, at page 11, paragraph [0043] to page 13,
paragraph [0053]. The FIX polypeptides described in International
Application No. PCT/US2011/043569 filed Jul. 11, 2011 and published
as WO 2012/006624 on Jan. 12, 2012 are also incorporated herein by
reference in its entirety.
[0083] In addition, hundreds of non-functional mutations in FIX
have been identified in hemophilia patients, many of which are
disclosed in Table 1, at pages 11-14 of International publication
number WO 09/137254 A2, which is herein incorporated by reference
in its entirety. Such non-functional mutations are not included in
the invention, but provide additional guidance for which mutations
are more or less likely to result in a functional FIX
polypeptide.
[0084] In one embodiment, the Factor IX (or Factor IX portion of a
chimeric polypeptide) may be at least 70%, at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100% identical to a FIX amino acid
sequence shown in Table 5A without a signal sequence and propeptide
sequence (amino acids 1 to 415 of SEQ ID NO:2), or alternatively,
with a propeptide sequence, or with a propeptide and signal
sequence (full-length FIX).
[0085] Factor IX coagulant activity is expressed as International
Unit(s) (IU). One IU of FIX activity corresponds approximately to
the quantity of FIX in one milliliter of normal human plasma.
Several assays are available for measuring Factor IX activity,
including the one stage clotting assay (activated partial
thromboplastin time; aPTT), thrombin generation time (TGA) and
rotational thromboelastometry (ROTEM.RTM.).
[0086] A chimeric polypeptide comprising a FIX polypeptide and an
FcRn binding partner can comprise an amino acid sequence at least
60%, at least 70%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identical to the Factor IX and FcRn BP, e.g., the Fc amino
acid sequence shown in Table 5A without a signal sequence and
propeptide sequence (amino acids 1 to 642 of SEQ ID NO:2), or
alternatively, with a propeptide sequence, or alternatively with a
signal sequence and a propeptide sequence.
[0087] A long-acting or long-lasting FIX polypeptide can be a
hybrid FIX polypeptide. Hybrid FIX polypeptide means a combination
of a FIX chimeric polypeptide with a second polypeptide. The
chimeric polypeptide and the second polypeptide in a hybrid may be
associated with each other via non-covalent protein-protein
interactions, such as charge-charge or hydrophobic interactions.
The chimeric polypeptide and the second polypeptide in a hybrid may
be associated with each other via covalent bond(s) such as
disulfide bonds. The chimeric peptide and the second peptide may be
associated with each other via more than one type of bond, such as
non-covalent and disulfide bonds. Hybrids are described in WO
2004/101740, WO2005/001025, U.S. Pat. No. 7,404,956, U.S. Pat. No.
7,348,004, and WO 2006/074199, each of which is incorporated herein
by reference in its entirety. The second polypeptide may be a
second copy of the same chimeric polypeptide or it may be a
non-identical chimeric polypeptide. In other embodiments, the
second polypeptide is a polypeptide comprising an FcRn BP, e.g.,
Fc. In some embodiments, the chimeric polypeptide is a Factor
IX-FcRn BP, e.g., Factor IX-Fc chimeric polypeptide, and the second
polypeptide consists essentially of Fc. See, e.g., Table 5A and B
(SEQ ID NOs:2 and 4). See, e.g., U.S. Pat. No. 7,404,956, which is
incorporated herein by reference in its entirety.
[0088] The second polypeptide in a hybrid may comprise or consist
essentially of a sequence at least 70%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% identical to the amino acid sequence
shown in Table 5B without a signal sequence (SEQ ID NO:4), or
alternatively, with a signal sequence.
[0089] In some embodiments, a long-acting FIX polypeptide is a FIX
monomer dimer hybrid. Monomer-dimer hybrid can comprise two
polypeptide chains, one chain comprising a FIX polypeptide and a
first Fc region, and another chain comprising, consisting
essentially of, or consisting of a second Fc region. In certain
aspects, a FIX monomer dimer hybrid consists essentially of or
consists of two polypeptide chains, a first chain consisting
essentially of or consisting of a FIX polypeptide and a second
chain consisting essentially of or consisting of a second Fc
region.
[0090] A long-acting FIX polypeptide can be encoded by a nucleotide
sequence which is at least 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to, for example, the nucleotide coding sequence in SEQ ID
NO:1 or 3 (the Factor IX portion, the Fc portion, individually or
together) or the complementary strand thereto, the nucleotide
coding sequence of known mutant and recombinant Factor IX or Fc
such as those disclosed in the publications and patents cited
herein or the complementary strand thereto, a nucleotide sequence
encoding the polypeptide of SEQ ID NO:2 or 4 (the Factor IX
portion, the Fc portion, individually or together), and/or
polynucleotide fragments of any of these nucleic acid molecules
(e.g., those fragments described herein). Polynucleotides which
hybridize to these nucleic acid molecules under stringent
hybridization conditions or lower stringency conditions are also
included as variants, as are polypeptides encoded by these
polynucleotides as long as they are functional.
[0091] B. Factor VIII
[0092] In some embodiments, a clotting factor for the invention is
a modified FVIII polypeptide. In one aspect, a modified FVIII
polypeptide is a long-acting FVIII polypeptide. In another aspect,
a long-acting FVIII polypeptide comprises a FVIII polypeptide and
an FcRn binding partner. The FVIII polypeptide means functional
factor VIII polypeptide in its normal role in coagulation, unless
otherwise specified. Thus, the term Factor VIII includes variant
polypeptides that are functional. Factor VIII proteins can be the
human, porcine, canine, and murine factor VIII proteins, in
addition, the full-length polypeptide and polynucleotide sequences
are known, as are many functional fragments, mutants and modified
versions. Examples of human factor VIII sequences are shown as
subsequences in SEQ ID NO: 6 or 8 (Table 7A and 7B). Factor VIII
polypeptides include, e.g., full-length factor VIII, fall-length
factor VIII minus Met at the N-terminus, mature factor VIII (minus
the signal sequence), mature factor VIII with an additional Met at
the N-terminus, and/or factor VIII with a full or partial deletion
of the B domain. Factor VIII variants include B domain deletions,
whether partial or full deletions.
[0093] A great many functional factor VIII variants are known, as
is discussed above and below. In addition, hundreds of
nonfunctional mutations in factor VIII have been identified in
hemophilia patients, and it has been determined that the effect of
these mutations on factor VIII function is due more to where they
lie within the 3-dimensional structure of factor VIII than on the
nature of the substitution (Cutler et al., Hum. Mutat. 19:274-8
(2002)), incorporated herein by reference in its entirety. In
addition, comparisons between factor VIII from humans and other
species have identified conserved residues that are likely to be
required for function (Cameron et al., Thromb. Haemost. 79:317-22
(1998); U.S. Pat. No. 6,251,632), incorporated herein by reference
in its entirety.
[0094] The human factor VIII gene was isolated and expressed in
mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984);
Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et
al., Nature 312:330-337 (1984); Vehar, G. A., et al., Nature
312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; U.S.
Pat. No. 4,757,006), each of which is incorporated herein by
reference in its entirety, and the amino acid sequence was deduced
from cDNA. Capon et al., U.S. Pat. No. 4,965,199, incorporated
herein by reference in its entirety, discloses a recombinant DNA
method for producing factor VIII in mammalian host cells and
purification of human factor VIII. Human factor VIII expression in
CHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney
cells) has been reported. Human factor VIII has been modified to
delete part or all of the B domain (U.S. Pat. Nos. 4,994,371 and
4,868,112, each of which is incorporated herein by reference in its
entirety), and replacement of the human factor VIII B domain with
the human factor V B domain has been performed (U.S. Pat. No.
5,004,803, incorporated herein by reference in its entirety). The
cDNA sequence encoding human factor VIII and predicted amino acid
sequence are shown in SEQ ID NO: 3B and 4B, respectively, of US
Application Publ. No. 2005/0100990, incorporated herein by
reference in its entirety.
[0095] U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein
by reference in its entirety, reports functional mutants of factor
VIII having reduced antigenicity and reduced immunoreactivity. U.S.
Pat. No. 6,376,463, Lollar, J. S., incorporated herein by reference
in its entirety, also reports mutants of factor VIII having reduced
immunoreactivity. US Application Publ. No. 2005/0100990, Saenko et
al., incorporated herein by reference in its entirety, reports
functional mutations in the A2 domain of factor VIII.
[0096] A number of functional factor VIII molecules, including
B-domain deletions, are disclosed in the following patents U.S.
Pat. No. 6,316,226 and U.S. Pat. No. 6,346,513, both assigned to
Baxter; U.S. Pat. No. 7,041,635 assigned to In2Gen; U.S. Pat. No.
5,789,203, U.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and
U.S. Pat. No. 6,228,620 assigned to Chiron; U.S. Pat. No. 5,972,885
and U.S. Pat. No. 6,048,720 assigned to Biovitrum, U.S. Pat. No.
5,543,502 and U.S. Pat. No. 5,610,278 assigned to Novo Nordisk;
U.S. Pat. No. 5,171,844 assigned to Immuno Ag; U.S. Pat. No.
5,112,950 assigned to Transgene S. A.; U.S. Pat. No. 4,868,112
assigned to Genetics Institute, each of which is incorporated
herein by reference in its entirety.
[0097] The porcine factor VIII sequence is published, (Toole, J.
J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)),
incorporated herein by reference in its entirety, and the complete
porcine cDNA sequence obtained from PCR amplification of factor
VIII sequences from a pig spleen cDNA library has been reported
(Healey, J. F. et al., Blood 88:4209-4214 (1996), incorporated
herein by reference in its entirety). Hybrid human/porcine factor
VIII having substitutions of all domains, all subunits, and
specific amino acid sequences were disclosed in U.S. Pat. No.
5,364,771 by Lollar and Runge, and in WO 93/20093, incorporated
herein by reference in its entirety. More recently, the nucleotide
and corresponding amino acid sequences of the A1 and A2 domains of
porcine factor VIII and a chimeric factor VIII with porcine A1
and/or A2 domains substituted for the corresponding human domains
were reported in WO 94/11503, incorporated herein by reference in
its entirety. U.S. Pat. No. 5,859,204, Lollar, J. S., also
discloses the porcine cDNA and deduced amino acid sequences. U.S.
Pat. No. 6,458,563, incorporated herein by reference in its
entirety assigned to Emory discloses a B-domain deleted porcine
Factor VIII.
[0098] The factor VIII (or Factor VIII portion of a chimeric
polypeptide) may be at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or at least 100% identical to a Factor VIII amino
acid sequence shown in Tables 7A and 7B without a signal sequence
(amino acids 20 to 1457 of SEQ ID NO: 6; and amino acids 20 to 2351
of SEQ ID NO: 8), wherein said Factor VIII portion has Factor VIII
activity. The Factor VIII (or Factor VII portion of a chimeric
polypeptide) may be identical to a Factor VIII amino acid sequence
shown in Tables 7A and 7B without a signal sequence (amino acids 20
to 1457 of SEQ ID NO: 6; and amino acids 20 to 2351 of SEQ ID NO:
8).
[0099] The Factor VIII (or Factor VIII portion of a chimeric
polypeptide) may be at least 60%, at least 70%, at least 80%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99%, or at least 100% identical to a Factor VIII amino
acid sequence shown in Tables 7A and 7B with a signal sequence
(amino acids 1 to 1457 of SEQ ID NO: 6 and amino acids 1 to 2351 of
SEQ ID NO: 8), wherein the Factor VIII portion has Factor VIII
activity. The Factor VIII (or Factor VIII portion of a chimeric
polypeptide) may be identical to a Factor VIII amino acid sequence
shown in Tables 7A and 7B with a signal sequence (amino acids 1 to
1457 of SEQ ID NO: 6 and amino acids 1 to 2351 of SEQ ID NO:
8).
[0100] A "B domain" of Factor VIII, as used herein, is the same as
the B domain known in the art that is defined by internal amino
acid sequence identity and sites of proteolytic cleavage by
thrombin, e.g., residues Ser741-Arg1648 of full-length human factor
VIII. The other human factor VIII domains are defined by the
following amino acid residues: A1, residues Ala1-Arg372; A2,
residues Ser373-Arg740; A3, residues Ser1690-Ile2032; C1, residues
Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2
sequence includes residues Ser1690-Tyr2332. The remaining sequence,
residues Glu1649-Arg1689, is usually referred to as the factor VIII
light chain activation peptide. The locations of the boundaries for
all of the domains, including the B domains, for porcine, mouse and
canine factor VIII are also known in the art. In one embodiment,
the B domain of Factor VIII is deleted ("B domain deleted factor
VIII" or "BDD FVIII"). An example of a BDD FVIII is REFACTO.RTM.
(recombinant SQ BDD FVIII), which has the same sequence as the
Factor VIII portion of the sequence in Table 7A (amino acids 1 to
1457 or 20 to 1457 of SEQ ID NO:6). In another embodiment, the B
domain deleted Factor VIII contains an intact intracellular
processing site, which corresponds to Arginine at residue 754 of B
domain deleted Factor VIII, which corresponds to Arginine residue
773 of SEQ ID NO: 6, or residue 1648 of full-length Factor VIII,
which corresponds to Arginine residue 1667 of SEQ ID NO: 8. The
sequence residue numbers used herein without referring to any SEQ
ID Numbers correspond to the Factor VIII sequence without the
signal peptide sequence (19 amino acids) unless otherwise
indicated. For example, S743/Q1638 of full-length Factor VIII
corresponds to S762/Q1657 of SEQ ID NO: 8 due to the 19 amino acid
signal peptide sequence. In other embodiments, the B domain deleted
FVIII comprises a substitution or mutation at an amino acid
position corresponding to Arginine 1645, a substitution or mutation
at an amino acid position corresponding to Arginine 1648, or a
substitution or mutation at amino acid positions corresponding to
Arginine 1645 and Arginine 1648 in full-length Factor VIII. In some
embodiments, the amino acid substituted at the amino acid position
corresponding to Arginine 1645 is a different amino acid from the
amino acid substituted at the amino acid position corresponding to
Arginine 1648. In certain embodiments, the substitution or mutation
is an amino acid other than arginine, e.g., alanine.
[0101] A "B domain deleted factor VIII" may have the full or
partial deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513,
7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885,
6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112,
and 6,458,563, each of which is incorporated herein by reference in
its entirety. In some embodiments, a B domain deleted factor VIII
sequence of the present invention comprises any one of the
deletions disclosed at col. 4, line 4 to col. 5, line 28 and
examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No.
6,346,513). In some embodiments, a B domain deleted factor VIII of
the present invention has a deletion disclosed at col. 2, lines
26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat.
No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No.
6,228,620). In some embodiments, a B domain deleted factor VIII has
a deletion described in col. 1, lines 25 to col. 2, line 40 of U.S.
Pat. No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat.
No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col.
4, line 22 to col. 5, line 36 of U.S. Pat. No. 5,171,844; col. 2,
lines 55-68, FIG. 2, and example 1 of U.S. Pat. No. 5,112,950; col.
2, line 2 to col. 19, line 21 and table 2 of U.S. Pat. No.
4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to
col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11,
line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4,
lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments, a B
domain deleted factor VIII has a deletion of most of the B domain,
but still contains amino-terminal sequences of the B domain that
are essential for in vivo proteolytic processing of the primary
translation product into two polypeptide chain (i.e., intracellular
processing site), as disclosed in WO 91/09122, which is
incorporated herein by reference in its entirety. In some
embodiments, a B domain deleted factor VIII is constructed with a
deletion of amino acids 747-1638, i.e., virtually a complete
deletion of the B domain. Hoeben R. C., et al. J, Biol. Chem. 265
(13): 7318-7323 (1990), incorporated herein by reference in its
entirety. A B domain deleted factor VIII may also contain a
deletion of amino acids 771-1666 or amino acids 868-1562 of factor
VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988),
incorporated herein by reference in its entirety. Additional B
domain deletions that are part of the invention include, e.g.:
deletion of amino acids 982 through 1562 or 760 through 1639 (Toole
et al., Proc. Natl. Acad. Sci. U.S.A. 83:5939-5942 (1986)), 797
through 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), 741
through 1646 (Kaufman (PCT published application No. WO 87/04187)),
747-1560 (Sarver et al., DNA 6:553-564 (1987)), 741 through 1648
(Pasek (PCT application No. 88/00831)), 816 through 1598 or 741
through 1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP
295597)), each of which is incorporated herein by reference in its
entirety. Each of the foregoing deletions may be made in any Factor
VIII sequence.
[0102] In one embodiment, the B domain deleted Factor VIII portion
in the chimeric polypeptide is processed into two chains connected
(or associated) by a metal bond, the first chain comprising a heavy
chain (A1-A2-partial B) and a second chain comprising a light chain
(A3-C1-C2). In another embodiment, the B domain deleted Factor VIII
portion is a single chain Factor VIII or unprocessed FVIII. The
single chain Factor VIII can comprise an intracellular processing
site, which corresponds to Arginine at residue 754 of B domain
deleted Factor VIII (i.e., residue 773 of SEQ ID NO: 6) or at
residue 1648 of full-length Factor VIII (i.e., residue 1657 of SEQ
ID NO: 8).
[0103] The metal bond between the heavy chain and the light chain
can be any metal known in the art. For example, the metals useful
for the invention can be a divalent metal ion. The metals that can
be used to associate the heavy chain and light chain include, but
not limited to, Ca2+, Mn2+, or Cu2+. Fatouros et al., Intern. J
Pharm. 155(1): 121-131 (1997); Wakabayashi et al., JBC. 279(13):
12677-12684 (2004).
[0104] In other embodiments, a FVIII polypeptide of the invention
is processed FVIII comprising a light chain and a heavy chain of
FVIII. In yet other embodiments, a FVIII polypeptide is single
chain FVIII. In other embodiments, the single chain FVIII comprises
a substitution or mutation at an amino acid position corresponding
to Arginine 1645, a substitution or mutation at an amino acid
position corresponding to Arginine 1648, or a substitution or
mutation at amino acid positions corresponding to Arginine 1645 and
Arginine 1648 in full-length Factor VIII. In some embodiments, the
amino acid substituted at the amino acid position corresponding to
Arginine 1645 is a different amino acid from the amino acid
substituted at the amino acid position corresponding to Arginine
1648. In certain embodiments, the substitution or mutation is an
amino acid other than arginine, e.g., alanine.
[0105] In one embodiment, a chimeric polypeptide comprising a FVIII
polypeptide and an FcRn binding partner can comprise an amino acid
sequence at least 70%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identical to the Factor VIII and FcRn BP, e.g., the Fc
amino acid sequence shown in Table 5B without a signal sequence
(SEQ ID NO:4).
[0106] In another embodiment, a chimeric polypeptide comprising a
FVIII polypeptide and an FcRn binding partner can comprise an amino
acid sequence at least 70%, at least 80%, at least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% identical to the Factor VIII and FcRn BP, e.g.,
the Fc amino acid sequence shown in Table 5B without a signal
sequence (SEQ ID NO:4).
[0107] A long-acting or long-lasting FVIII polypeptide can be a
hybrid FVIII polypeptide. Hybrid FVIII polypeptide means a
combination of a FVIII chimeric polypeptide with a second
polypeptide. The chimeric polypeptide and the second polypeptide in
a hybrid may be associated with each other via non-covalent
protein-protein interactions, such as charge-charge or hydrophobic
interactions. The chimeric polypeptide and the second polypeptide
in a hybrid may be associated with each other via covalent bond(s)
such as disulfide bonds. The chimeric peptide and the second
peptide may be associated with each other via more than one type of
bond, such as non-covalent and disulfide bonds. The second
polypeptide may be a second copy of the same chimeric polypeptide
or it may be a non-identical chimeric polypeptide. In other
embodiments, the second polypeptide is a polypeptide comprising an
FcRn BP, e.g., Fc. In some embodiments, the chimeric polypeptide is
a Factor VIII-FcRn BP, e.g., Factor VIII-Fc chimeric polypeptide,
and the second polypeptide consists essentially of Fc.
[0108] The second polypeptide in a hybrid may comprise or consist
essentially of a sequence at least 70%, at least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or 100% identical to the amino acid sequence
shown in Table 5B without a signal sequence (SEQ ID NO:4), or
alternatively, with a signal sequence.
[0109] In some embodiments, a long-acting FVIII polypeptide is a
FVIII monomer dimer hybrid. Monomer-dimer hybrids can comprise two
polypeptide chains, one chain comprising a FVIII polypeptide and a
first Fc region, and another chain comprising, consisting
essentially of, or consisting of a second Fc region. In certain
aspects, a FVIII monomer dimer hybrid consists essentially of or
consists of two polypeptide chains, a first chain consisting
essentially of or consisting of a FVIII polypeptide and a second
chain consisting essentially of or consisting of a second Fc
region.
[0110] In some embodiments, a long-acting FVIII polypeptide
comprises a FVIII polypeptide and at least one heterologous moiety,
which increases in vivo half-life of the FVIII polypeptide, wherein
the at least one heterologous moiety is linked to the C-terminus or
N-terminus of the FVIII polypeptide or inserted between two amino
acids of the FVIII polypeptide.
[0111] A long-acting FVIII polypeptide can be encoded by a
nucleotide sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%
or 99% identical to, for example, the nucleotide coding sequence in
SEQ ID NO:5 or 7 (the Factor VIII portion, the Fc portion,
individually or together) or the complementary strand thereto, the
nucleotide coding sequence of known mutant and recombinant Factor
VIII or Fc such as those disclosed in the publications and patents
cited herein or the complementary strand thereto, a nucleotide
sequence encoding the polypeptide of SEQ ID NO:6 or 8 (the Factor
VIII portion, the Fc portion, individually or together), and/or
polynucleotide fragments of any of these nucleic acid molecules
(e.g., those fragments described herein). Polynucleotides which
hybridize to these nucleic acid molecules under stringent
hybridization conditions or lower stringency conditions are also
included as variants, as are polypeptides encoded by these
polynucleotides as long as they are functional.
[0112] By a nucleic acid having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence, it is
intended that the nucleotide sequence of the nucleic acid is
identical to the reference sequence except that the nucleotide
sequence may include up to five point mutations per each 100
nucleotides of the reference nucleotide sequence. In other words,
to obtain a nucleic acid having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. The query sequence may be, for example, the
entire sequence shown in SEQ ID NO:1 or 3, the ORF (open reading
frame), or any fragment specified as described herein.
[0113] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to a nucleotide sequence or polypeptide of the
present invention can be determined conventionally using known
computer programs. In one embodiment, the best overall match
between a query sequence (reference or original sequence) and a
subject sequence, also referred to as a global sequence alignment,
can be determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245),
which is herein incorporated by reference in its entirety. In a
sequence alignment the query and subject sequences are both DNA
sequences. An RNA sequence can be compared by converting U's to
T's. The result of said global sequence alignment is in percent
identity. In certain embodiments, the parameters used in a FASTDB
alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject
nucleotide sequence, whichever is shorter.
[0114] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0115] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0116] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, or substituted with another
amino acid. These alterations of the reference sequence may occur
at the amino or carboxy terminal positions of the reference amino
acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0117] As a practical matter, whether any particular polypeptide is
at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for
instance, the amino acid sequences of SEQ ID NOs:2, 4, 6, or 8 (the
FIX portion, the FVIII portion, the Fc portion, individually or
together), or a known FIX, FVIII, or Fc polypeptide sequence, can
be determined conventionally using known computer programs. In one
embodiment, the best overall match between a query sequence
(reference or original sequence) and a subject sequence, also
referred to as a global sequence alignment, can be determined using
the FASTDB computer program based on the algorithm of Brutlag et
al., Comp. App. Biosci. 6:237-245(1990), incorporated herein by
reference in its entirety. In a sequence alignment the query and
subject sequences are either both nucleotide sequences or both
amino acid sequences. The result of said global sequence alignment
is in percent identity. In certain embodiments, the parameters used
in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,
Mismatch Penalty=1, Joining Penalty=20, Randomization Group
Length=0, Cutoff Score=1, Window Size-sequence length, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of
the subject amino acid sequence, whichever is shorter.
[0118] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention, Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0119] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0120] The polynucleotide variants may contain alterations in the
coding regions, non-coding regions, or both. Certain embodiments
include polynucleotide variants containing alterations which
produce silent substitutions, additions, or deletions, but do not
alter the properties or activities of the encoded polypeptide.
Nucleotide variants can be produced by silent substitutions due to
the degeneracy of the genetic code. Moreover, variants in which
5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in
any combination are included. Polynucleotide variants can be
produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the human mRNA
to those preferred by a bacterial host such as E. coli).
[0121] Naturally occurring variants are called "allelic variants,"
and refer to one of several alternate forms of a gene occupying a
given locus on a chromosome of an organism (Genes II, Lewin, B.,
ed., John Wiley & Sons, New York (1985)). These allelic
variants can vary at either the polynucleotide and/or polypeptide
level and are included in the present invention. Alternatively,
non-naturally occurring variants may be produced by mutagenesis
techniques or by direct synthesis.
[0122] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides. For instance, one or more
amino acids can be deleted from the N-terminus or C-terminus of the
secreted protein without substantial loss of biological function.
The authors of Ron et al., J Biol. Chem. 268: 2984-2988 (1993),
incorporated herein by reference in its entirety, reported variant
KGF proteins having heparin binding activity even after deleting 3,
8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma exhibited up to ten times higher activity after deleting 8-10
amino acid residues from the carboxy terminus of this protein.
(Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated
herein by reference in its entirety.)
[0123] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J Biol. Chem.
268:22105-22111 (1993), incorporated herein by reference in its
entirety) conducted extensive mutational analysis of human cytokine
IL-1a. They used random mutagenesis to generate over 3,500
individual IL-la mutants that averaged 2.5 amino acid changes per
variant over the entire length of the molecule. Multiple mutations
were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]."
(See Abstract.) In fact, only 23 unique amino acid sequences, out
of more than 3,500 nucleotide sequences examined, produced a
protein that significantly differed in activity from wild type.
[0124] As stated above, polypeptide variants include modified
polypeptides. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0125] C. Half-Life Extension
[0126] In certain aspects, a modified clotting factor of the
invention comprises at least one heterologous moiety which
increases the in vivo half-life of the protein. In vivo half-life
of a modified clotting factor can be determined by any method known
to those of skill in the art, e.g., clotting activity assays
(chromogenic assay or one stage clotting aPTT assay) to detect
plasma clotting activity levels or ELISA to detect plasma clotting
factor antigen level.
[0127] In certain aspects, a heterologous moiety which increases in
vivo half-life of the modified clotting factor of the invention can
comprise, without limitation, a heterologous polypeptide such as
albumin, an immunoglobulin Fc region, the .beta. subunit of the
C-terminal peptide (CTP) of human chorionic gonadotropin, a PAS
sequence, a HAP sequence, a transferrin, albumin-binding moieties,
or any fragments, derivatives, variants, or combinations of these
polypeptides. In certain aspects the modified clotting factor of
the invention comprises a heterologous polypeptide which increases
in vivo half-life. In other related aspects a heterologous moiety
can include an attachment site for a non-polypeptide moiety such as
polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic
acid, or any derivatives, variants, or combinations of these
elements.
[0128] In other embodiments, a modified clotting factor of the
invention is conjugated to one or more polymers. The polymer can be
water-soluble or non-water-soluble. The polymer can be covalently
or non-covalently attached to a clotting factor or to other
moieties conjugated to a clotting factor. Non-limiting examples of
the polymer can be poly(alkylene oxide), poly(vinyl pyrrolidone),
poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine).
Additional types of polymer-conjugated clotting factor are
disclosed in U.S. Pat. No. 7,199,223, which is disclosed by
reference in its entirety.
[0129] In certain aspects, a modified clotting factor of the
invention can comprise one, two, three or more heterologous
moieties, which can each be the same or different molecules.
[0130] D. FcRn Binding Partner
[0131] FcRn binding partner ("FcRn BP") comprises functional
neonatal Fc receptor (FcRn) binding partners, unless otherwise
specified. An FcRn binding partner is any molecule that can be
specifically bound by the FcRn receptor with consequent active
transport by the FcRn receptor of the FcRn binding partner. Thus,
the term FcRn BP includes any variants of IgG Fc that are
functional. For example, the region of the Fc portion of IgG that
binds to the FcRn receptor has been described based on X-ray
crystallography (Burmeister et al. 1994, Nature 372:379,
incorporated herein by reference in its entirety). The major
contact area of the Fc with the FcRn is near the junction of the
CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig
heavy chain. FcRn BPs include whole IgG, the Fc fragment of IgG,
and other fragments of IgG that include the complete binding region
of FcRn. The major contact sites include amino acid residues 248,
250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain
and amino acid residues 385-387, 428, and 433-436 of the CH3
domain. References made to amino acid numbering of immunoglobulins
or immunoglobulin fragments, or regions, are all based on Kabat et
al. 1991, Sequences of Proteins of Immunological interest, U. S.
Department of Public Health, Bethesda; MD, incorporated herein by
reference in its entirety. (The FcRn receptor has been isolated
from several mammalian species including humans. The sequences of
the human FcRn, rat FcRn, and mouse FcRn are known (Story et al.
1994, J. Exp. Med. 180: 2377), incorporated herein by reference in
its entirety.) An FcRn BP may comprise the CH2 and CH3 domains of
an immunoglobulin with or without the hinge region of the
immunoglobulin. In a particular embodiment, an FcRn BP is an Fc
region. Exemplary FcRn BP variants are provided in WO 2004/101740
and WO 2006/074199, incorporated herein by reference in its
entirety.
[0132] FcRn BP (or FcRn BP portion of a chimeric polypeptide) may
contain one or more mutations, and combinations of mutations.
[0133] FcRn BP (or FcRn BP portion of a chimeric polypeptide) may
contain mutations conferring increased half-life such as M252Y,
S254T, T256E, and combinations thereof, as disclosed in Oganesyan
et al., Mol. Immunol. 46:1750 (2009), which is incorporated herein
by reference in its entirety; H433K, N434F, and combinations
thereof, as disclosed in Vaccaro et al., Nat. Biotechnol. 23:1283
(2005), which is incorporated herein by reference in its entirety;
the mutants disclosed at pages 1-2, paragraph [0012], and Examples
9 and 10 of U.S. 2009/0264627 A1, which is incorporated herein by
reference in its entirety; and the mutants disclosed at page 2,
paragraphs [0014] to [0021] of U.S. 20090163699 A1, which is
incorporated herein by reference in its entirety.
[0134] FcRn BP (or FcRn BP portion of a chimeric polypeptide) may
also include the following mutations: The Fc region of IgG can be
modified according to well recognized procedures such as site
directed mutagenesis and the like to yield modified IgG or Fc
fragments or portions thereof that will be bound by FcRn. Such
modifications include modifications remote from the FcRn contact
sites as well as modifications within the contact sites that
preserve or even enhance binding to the FcRn. For example the
following single amino acid residues in human IgG1 Fc (Fcy1) can be
substituted without significant loss of Fc binding affinity for
FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A,
T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A,
Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A,
E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A,
V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A,
K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P331A, P331S,
E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A,
Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A,
S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A,
N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A,
R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A,
Q438A, K439A, S440A, S444A, and K447A, where for example P238A
represents wild type proline substituted by alanine at position
number 238. In addition to alanine other amino acids may be
substituted for the wild type amino acids at the positions
specified above. Mutations may be introduced singly into Fc giving
rise to more than one hundred FcRn binding partners distinct from
native Fc. Additionally, combinations of two, three, or more of
these individual mutations may be introduced together, giving rise
to hundreds more FcRn binding partners. Certain of these mutations
may confer new functionality upon the FcRn binding partner. For
example, one embodiment incorporates N297A, removing a highly
conserved N-glycosylation site. The effect of this mutation is to
reduce immunogenicity, thereby enhancing circulating half-life of
the FcRn binding partner, and to render the FcRn binding partner
incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA,
without compromising affinity for FcRn (Routledge et al. 1995,
Transplantation 60:847, which is incorporated herein by reference
in its entirety; Friend et al. 1999, Transplantation 68:1632, which
is incorporated herein by reference in its entirety; Shields et al.
1995, J. Biol. Chem. 276:6591, which is incorporated herein by
reference in its entirety). Additionally, at least three human Fc
gamma receptors appear to recognize a binding site on IgG within
the lower hinge region, generally amino acids 234-237. Therefore,
another example of new functionality and potential decreased
immunogenicity may arise from mutations of this region, as for
example by replacing amino acids 233-236 of human IgG1 "ELLG" to
the corresponding sequence from IgG2 "PVA" (with one amino acid
deletion). It has been shown that FcyRI, FcyRII, and FcyRIII which
mediate various effector functions will not bind to IgG1 when such
mutations have been introduced (Ward and Ghetie 1995, Therapeutic
Immunology 2:77, which is incorporated herein by reference in its
entirety; and Armour et al. 1999, Eur. J. Immunol. 29:2613, which
is incorporated herein by reference in its entirety). As a further
example of new functionality arising from mutations described
above, affinity for FcRn may be increased beyond that of wild type
in some instances. This increased affinity may reflect an increased
"on" rate, a decreased "off" rate or both an increased "on" rate
and a decreased "off" rate. Mutations believed to impart an
increased affinity for FcRn include T256A, T307A, E380A, and N434A
(Shields et al. 2001, J. Biol. Chem. 276:6591, which is
incorporated herein by reference in its entirety).
[0135] The FcRn BP (or FcRn BP portion of a chimeric polypeptide)
may be at least 70%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
or 100% identical to the Fc amino acid sequence shown in Table 5B
without a signal sequence (SEQ ID NO:4), or alternatively, with a
signal sequence.
[0136] Myriad mutants, fragments, variants, and derivatives are
described, e.g., in PCT Publication Nos. WO 2011/069164 A2, WO
2012/006623 A2, WO 2012/006635 A2, or WO 2012/006633 A2, all of
which are incorporated herein by reference in their entireties.
[0137] E. Albumins
[0138] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one albumin
polypeptide or fragment, variant, or derivative thereof, wherein
the modified clotting factor has procoagulant activity and can be
expressed in vivo or in vitro in a host cell. Human serum albumin
(HSA, or HA), a protein of 609 amino acids in its full-length form,
is responsible for a significant proportion of the osmotic pressure
of serum and also functions as a carrier of endogenous and
exogenous ligands. The term "albumin" as used herein includes
full-length albumin or a functional fragment, variant, derivative,
or analog thereof. Examples of albumin or the fragments or variants
thereof are disclosed in US Pat. Publ. Nos. 2008/0194481A1,
2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or 2008/0153751
A1 or PCT Appl. Publ. Nos. 2008/033413 A2, 2009/058322 A1, or
2007/021494 A2, which are incorporated herein by reference in their
entireties.
[0139] The albumin binding polypeptides can compromise, without
limitation, bacterial albumin-binding domains, albumin-binding
peptides, or albumin-binding antibody fragments that can bind to
albumin. Domain 3 from streptococcal protein G, as disclosed by
Kraulis et al., FEBS Lett. 378:190-194 (1996) and Linhult et al.,
Protein Sci. 11:206-213 (2002) is an example of a bacterial
albumin-binding domain. Examples of albumin-binding peptides
include a series of peptides having the core sequence DICLPRWGCLW
(SEQ ID NO:18). See, e.g., Dennis et al., J. Biol. Chem. 2002, 277:
35035-35043 (2002). Examples of albumin-binding antibody fragments
are disclosed in Muller and Kontermann, Curr. Opin. Mol. Ther.
9:319-326 (2007); Roovers et al., Cancer Immunol. Immunother.
56:303-317 (2007), and Holt et al., Prot. Eng. Design Sci.,
21:283-288 (2008), which are incorporated herein by reference in
their entireties.
[0140] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one attachment
site for a non-polypeptide small molecule, variant, or derivative
that can bind to albumin thereof. For example, a modified clotting
factor of the invention can include one or more organic albumin
binding moieties attached to the clotting factor. An example of
such albumin binding moieties is
2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate
("Albu" tag) as disclosed by Trussel et al., Bioconjugate Chem.
20:2286-2292 (2009).
[0141] F. CTP
[0142] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one C-terminal
peptide (CTP) of the .beta. subunit of human chorionic gonadotropin
or fragment, variant, or derivative thereof. One or more CTP
peptides fused to or inserted into a clotting factor is known to
increase the in vivo half-life of that protein. See, e.g., U.S.
Pat. No. 5,712,122, incorporated by reference herein in its
entirety. Exemplary CTP peptides include
DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO:9) or
SSSSKAPPPSLPSPSRLPGPSDTPILPQ. (SEQ ID NO:10). See, e.g., U.S.
Patent Application Publication No. US 2009/0087411 A1, incorporated
by reference.
[0143] G. PAS
[0144] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one PAS peptide
or fragment, variant, or derivative thereof. A PAS peptide or PAS
sequence, as used herein, means an amino acid sequence comprising
mainly alanine and serine residues or comprising mainly alanine,
serine, and proline residues, the amino acid sequence forming
random coil conformation under physiological conditions.
Accordingly, the PAS sequence is a building block, an amino acid
polymer, or a sequence cassette comprising, consisting essentially
of, or consisting of alanine, serine, and proline which can be used
as a part of the heterologous moiety in the chimeric protein. An
amino acid polymer also can form random coil conformation when
residues other than alanine, serine, and proline are added as a
minor constituent in the PAS sequence. By "minor constituent" is
meant that that amino acids other than alanine, serine, and proline
can be added in the PAS sequence to a certain degree, e.g., up to
about 12%, i.e., about 12 of 100 amino acids of the PAS sequence,
up to about 10%, up to about 9%, up to about 8%, about 6%, about
5%, about 4%, about 3%, i.e. about 2%, or about 1%, of the amino
acids. The amino acids different from alanine, serine and proline
cab be selected from the group consisting of Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and
Val. Under physiological conditions, a PAS peptide forms a random
coil conformation and thereby can mediate an increased in vivo
and/or in vitro stability to a recombinant protein of the
invention, and has procoaguiant activity.
[0145] Non-limiting examples of the PAS peptides include
ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 11), AAPASPAPAAPSAPAPAAPS (SEQ ID
NO:12), APSSSPSPSAPSSPSPASPSS (SEQ ID NO:13), APSSPSPSAPSSPSPASPS
(SEQ ID NO:14), SSPSAPSPSSPASPSPSSPA (SEQ ID) NO:15),
AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO:16), ASAAAPAAASAAASAPSAAA (SEQ
ID NO:17) or any variants, derivatives, fragments, or combinations
thereof. Additional examples of PAS sequences are known from, e.g.,
US Pat. Publ. No. 2010/0292130 A1 and PCT Appl. Publ. No. WO
2008/155134 A1. European issued patent EP2173890.
[0146] H. HAP
[0147] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one homo-amino
acid polymer (HAP) peptide or fragment, variant, or derivative
thereof. A HAP peptide can comprise a repetitive sequence of
glycine, which has at least 50 amino acids, at least 100 amino
acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino
acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino
acids, 400 amino acids, 450 amino acids, or 500 amino acids in
length. A HAP sequence is capable of extending half-life of a
moiety fused to or linked to the HAP sequence. Non-limiting
examples of the HAP sequence includes, but are not limited to
(Gly), (Gly.sub.4Ser).sub.n or S(Gly.sub.4Ser).sub.n, wherein n is
1, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20. In one embodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another
embodiment, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, or 200. See, e.g., Schlapschy M et at, Protein
Eng. Design Selection, 20: 273-284 (2007).
[0148] I. Transferrin
[0149] In certain aspects, a modified clotting factor of the
invention comprises at least one transferrin peptide or fragment,
variant, or derivative thereof linked to or inserted into the
clotting factor, wherein the modified clotting factor has
procoagulant activity. Any transferrin can be linked to or inserted
into a modified clotting factor of the invention. As an example,
wild-type human Tf (Tf) is a 679 amino acid protein, of
approximately 75 KDa (not accounting for glycosylation), with two
main domains, N (about 330 amino acids) and C (about 340 amino
acids), which appear to originate from a gene duplication. See
GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM
039847 and S95936 (www.ncbi.nlm.nih.gov), all of which are herein
incorporated by reference in their entirety.
[0150] Transferrin transports iron through transferrin receptor
(TfR)-mediated endocytosis. After the iron is released into an
endosomal compartment and Tf-TfR complex is recycled to cell
surface, the Tf is released back extracellular space for next cycle
of iron transporting. Tf possesses a long half-life that is in
excess of 14-17 days (Li et al., Trends Pharmacol. Sci. 23:206-209
(2002)). Transferrin fusion proteins have been studied for
half-life extension, targeted deliver for cancer therapies, oral
delivery and sustained activation of proinsulin (Brandsma et al.,
Biotechnol. Adv., 29: 230-238 (2011); Bai et al., Proc. Natl. Acad.
Sci. USA 102:7292-7296 (2005); Kim et al., J. Pharmacol. Exp.
Ther., 334:682-692 (2010); Wang et al., J. Controlled Release
155:386-392 (2011)).
[0151] J. PEG
[0152] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one attachment
site for a non-polypeptide heterologous moiety or fragment,
variant, or derivative thereof linked to or inserted into the
clotting factor, wherein the modified clotting factor has
procoagulant activity. For example, a modified clotting factor of
the invention can include one or more polyethylene glycol (PEG)
moieties attached to or inserted into the clotting factor, wherein
the modified clotting factor has procoagulant activity.
[0153] PEGylated clotting factor can refer to a conjugate formed
between clotting factor and at least one polyethylene glycol (PEG)
molecule. PEG is commercially available in a large variety of
molecular weights and average molecular weight ranges. Typical
examples of PEG average molecular weight ranges include, but are
not limited to, about 200, about 300, about 400, about 600, about
1000, about 1300-1600, about 1450, about 2000, about 3000, about
3000-3750, about 3350, about 3000-7000, about 3500-4500, about
5000-7000, about 7000-9000, about 8000, about 10000, about
8500-11500, about 16000-24000, about 35000, about 40000, about
60000, and about 80000 daltons. These average molecular weights are
provided merely as examples and are not meant to be limiting in any
way.
[0154] A modified clotting factor of the invention can be PEGylated
to include mono- or poly- (e.g., 2-4) PEG moieties. PEGylation can
be carried out by any of the PEGylation reactions known in the art.
Methods for preparing a PEGylated protein product will generally
include (i) reacting a polypeptide with polyethylene glycol (such
as a reactive ester or aldehyde derivative of PEG) under conditions
whereby the peptide of the invention becomes attached to one or
more PEG groups; and (ii) obtaining the reaction product(s). In
general, the optimal reaction conditions for the reactions will be
determined case by case based on known parameters and the desired
result.
[0155] There are a number of PEG attachment methods available to
those skilled in the art, for example Malik F et al., Exp. Hematol.
20:1028-35 (1992); Francis, Focus on Growth Factors 3(2):4-10
(1992); European Pat. Pub. Nos. EP0401384, EP0154316, and
EP0401384; and International Pat. Appl. Pub. Nos. WO92/16221 and
WO95/34326. As a non-limiting example, clotting factor variants can
contain cysteine substitutions in one or more permissive loops as
described herein, and the cysteines can be further conjugated to
PEG polymer. See Mei et al., Blood 116:270-279 (2010) and U.S. Pat.
No. 7,632,921, which are incorporated herein by reference in their
entireties.
[0156] K. HES
[0157] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one hydroxyethyl
starch (HES) polymer conjugated to or inserted into the clotting
factor, wherein the modified clotting factor has procoagulant
activity. HES is a derivative of naturally occurring amylopectin
and is degraded by alpha-amylase in the body. HES exhibits
advantageous biological properties and is used as a blood volume
replacement agent and in hemodilution therapy in the clinics. See,
e.g., Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987);
and Weidler et al., Arzneim.-Forschung/Drug Res. 41: 494-498
(1991).
[0158] HES is mainly characterized by the molecular weight
distribution and the degree of substitution. HES has a mean
molecular weight (weight mean) of from 1 to 300 kD, from 2 to 200
kD, from 3 to 100 kD, or from 4 to 70 kD. Hydroxyethyl starch can
further exhibit a molar degree of substitution of from 0.1 to 3,
from 0.1 to 2, from 0.1 to 0.9, or from 0.1 to 0.8, and a ratio
between C2:C6 substitution in the range of from 2 to 20 with
respect to the hydroxyethyl groups. HES with a mean molecular
weight of about 130 kD is VOLUVEN.RTM. from Fresenius. VOLUVEN.RTM.
is an artificial colloid, employed, e.g., for volume replacement
used in the therapeutic indication for therapy and prophylaxis of
hypovolaemia. There are a number of HES attachment methods
available to those skilled in the art, e.g., the same PEG
attachment methods described above.
[0159] L. PSA
[0160] In certain aspects, a modified clotting factor of the
invention comprises a clotting factor and at least one polysialic
acid (PSA) polymer conjugated to or inserted into the clotting
factor, wherein the modified clotting factor has procoagulant
activity. PSAs are naturally occurring unbranched polymers of
sialic acid produced by certain bacterial strains and in mammals in
certain cells. See, e.g., Roth J. et al. (1993) in Polysialic Acid:
From Microbes to Man, eds. Roth J., Rutishauser U., Troy F. A.
(BirkhauserVerlag, Basel, Switzerland), pp. 335-348. PSAs can be
produced in various degrees of polymerization from n=about 80 or
more sialic acid residues down to n=2 by limited acid hydrolysis or
by digestion with neuraminidases, or by fractionation of the
natural, bacterially derived forms of the polymer. There are a
number of PSA attachment methods available to those skilled in the
art, e.g., the same PEG attachment methods described above. In
certain aspects, an activated PSA can also be attached to a
cysteine amino acid residue on the clotting factor. See, e.g., U.S.
Pat. No. 5,846,951.
[0161] M. Clearance Receptors
[0162] In certain aspects, the in vivo half-life of a modified
clotting factor of the invention can be extended where the modified
clotting factor comprises at least one fragment of a clotting
factor clearance receptor or fragment, variant, or derivative
thereof linked to or inserted into the clotting factor, wherein the
modified clotting factor has procoagulant activity. For example,
insertion of soluble forms of clearance receptors, such as the low
density lipoprotein-related protein receptor LRP1, or fragments
thereof, can block binding of FVII to clearance receptors and
thereby extend its in vivo half-life. LRP1 is a 600 kDa integral
membrane protein that is implicated in the receptor-mediate
clearance of a variety of proteins, including FVIII. See, e.g.,
Lenting et al., Haemophilia 16:6-16 (2010). Other suitable FVIII
clearance receptors are, e.g., LDLR (low-density lipoprotein
receptor), VLDLR (very low-density lipoprotein receptor), and
megalin (LRP-2), or fragments thereof. See, e.g., Bovenschen et
al., Blood 106:906-912 (2005); Bovenschen, Blood 116:5439-5440
(2010); Martinelli et al., Blood 116:5688-5697 (2010).
III. DOSING STRATEGIES FOR CLOTTING FACTORS
[0163] The present invention provides a dosing strategy for a
clotting factor. A good dosing strategy provides reduced
interpatient variability in pharmacokinetics and pharmacodynamics.
While clotting factors have routinely been dosed based on the body
weight of the patient, the present invention shows that a fixed
dosing regimen is suitable for clotting factors that have a wide
therapeutic window.
[0164] In one aspect, the invention provides methods of
administering a clotting factor to a subject in need thereof,
comprising administering to the subject a fixed dose of a clotting
factor. Administration of the clotting factor is a replacement
therapy by providing a recombinant clotting factor to a subject
with clotting factor deficiency. Administration of the clotting
factor can reduce the number of bleeding episodes or prevent the
symptoms of a bleeding disorder in the subject.
[0165] In another aspect, the invention provides a method of
reducing, ameliorating, or preventing one or more symptoms of a
bleeding disease or disorder in a subject comprising administering
a fixed dose of a clotting factor to the subject in need thereof.
The invention also provides use of a fixed dose of a clotting
factor for the manufacture of a medicament for reducing,
ameliorating, or preventing one or more symptoms of a bleeding
disease or disorder in a subject in need thereof. The one or more
symptoms of a bleeding disease or disorder can be one or more
bleeding episodes. The bleeding episodes can be spontaneous or
caused by trauma or surgery. The invention can control bleeding or
prevent one or more bleeding episodes. The subject can be bleeding
at the time of administration or be expected to be bleeding, or can
be susceptible to bleeding as the result of minor hemorrhage,
hemarthroses, superficial muscle hemorrhage, soft tissue
hemorrhage, moderate hemorrhage, intramuscle or soft tissue
hemorrhage with dissection, mucous membrane hemorrhage, hematuria,
major hemorrhage, hemorrhage of the pharynx, hemorrhage of the
retropharynx, hemorrhage of the retroperitonium, hemorrhage of the
central nervous system, bruises, cuts, scrapes, joint hemorrhage,
nose bleed, mouth bleed, gum bleed, intracranial bleeding,
intraperitoneal bleeding, minor spontaneous hemorrhage, bleeding
after major trauma, moderate skin bruising, or spontaneous
hemorrhage into joints, muscles, internal organs or the brain. Such
subjects also include those in need of peri-operative management,
such as management of bleeding associated with surgery or dental
extraction. In one aspect, the subject is in need of prophylaxis of
one or more bleeding episodes. In another aspect, the subject is in
need of individualized interval prophylaxis. In other aspects, the
subject is in need of on-demand treatment or episodic treatment of
one or more bleeding episodes. In still other aspects, the subject
is in need of perioperative management of one or more bleeding
episodes.
[0166] In other aspects, the invention includes a method of
manufacturing a pharmaceutical composition, or compositions
comprising formulating a fixed dose of a clotting factor. The fixed
dose manufactured by the present method can be administered to a
subject in need thereof. The pharmaceutical composition(s) can
comprise, consist essentially or, or consist of a fixed dose of a
clotting factor and one or more pharmaceutically acceptable carrier
or excipient, but does not comprise any additional amount of the
clotting factor. In some embodiments, the entire fixed dose is
administered to the subject, i.e., no portion of the composition is
left unused.
[0167] In some aspects, the invention provides a pharmaceutical
composition comprising a fixed dose of a clotting factor and a
pharmaceutically acceptable carrier for use to reduce, ameliorate,
or prevent one or more symptoms of a bleeding disease or disorder
to a subject in need thereof. The pharmaceutical composition can
comprise, consist essentially or, or consist of a fixed dose of a
clotting factor and one or more pharmaceutically acceptable carrier
or excipient, but does not comprise any additional amount of the
clotting factor. In some embodiments, the entire fixed dose is
administered to the subject, i.e., no portion of the composition is
left unused. In certain embodiments, the pharmaceutical composition
comprises a fixed dose of a clotting factor, wherein the fixed dose
is provided in two or more (e.g., two, three, four, or five) vials.
The total contents of which provide the fixed dosage of the
clotting factor.
[0168] A clotting factor can be formulated as a pharmaceutical
composition. The pharmaceutical composition can be formulated for
administration to humans. The pharmaceutical compositions used in
the methods of this invention comprise pharmaceutically acceptable
carriers, including, e.g., ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat. Various methods of formulating the invention
are well known in the art.
[0169] In still other aspects, the invention is directed to a kit
for administration of a fixed dose amount of a clotting factor
comprising one or more containers (e.g., vials) of a pharmaceutical
composition and an instructional material. In one embodiment, a kit
comprises a single vial of a pharmaceutical composition comprising
a fixed dose of a clotting factor and an instructional material,
wherein the composition in the single vial is to be administered in
its entirety to a subject in need thereof. The instruction material
that can be inserted in the kit can comprise instructions to
administer the pharmaceutical composition of the clotting factor to
the subject. In another embodiment, a kit comprises an x number of
the pharmaceutical compositions, wherein x is any integer, and an
instructional material, wherein each of the pharmaceutical
compositions, e.g., each vial, comprises a portion of a clotting
factor, wherein the total amount of the clotting factor, when
combined, is a fixed dose for a subject in need thereof. For
example, x can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. When the kit
comprises two or more (i.e., x=2 or more) pharmaceutical
compositions (e.g., vials), each comprising a portion of a clotting
factor, the two or more compositions can be combined together into
one vial or syringe. Techniques for combining vials, e.g., by using
a large syringe, are known in the art. The combination of the
portions of the two or more compositions provides the fixed dose of
the clotting factor that is to be administered to the subject in
need thereof. In some embodiments, the entire fixed dose is
administered to the subject, i.e., no portion of the composition is
left unused.
[0170] In one example, a kit for administration of a fixed dose
amount of a clotting factor (e.g., a fixed dose of 4000 IU)
comprises a first container of a pharmaceutical composition and a
second container of a pharmaceutical composition, wherein the first
container comprises a first portion of a fixed dose of a clotting
factor (e.g., a vial containing 2,000 IU of the clotting factor)
and the second container comprises a second portion of the fixed
dose of the clotting factor (e.g., a second vial containing 2,000
IU of the clotting factor) and wherein the total amount of the
first container and the second container, when combined, is the
fixed dose (e.g., 4,000 IU). In another example, a kit for
administration of a fixed dose amount of a clotting factor (e.g., a
fixed dose of 6000 IU) comprises a first container of a
pharmaceutical composition, a second container of a pharmaceutical
composition, and a third container of a pharmaceutical composition,
wherein the first container comprises a first portion of a fixed
dose of a modified clotting factor (e.g., a vial containing 2,000
IU of the clotting factor), the second container comprises a second
portion of the fixed dose of the clotting factor (e.g., a second
vial containing 2,000 IU of the clotting factor), and the third
container comprises a third portion of the fixed dose of the
clotting factor (e.g., a third vial containing 2,000 IU of the
clotting factor) and wherein the total amount of the first
container, the second container, and the third container is the
fixed dose (e.g., 6,000 IU). In other examples, the first portion
of the first pharmaceutical composition, the second portion of the
pharmaceutical composition, and the third portion of the
pharmaceutical composition are the same or different. The
combination of the first and second composition (i.e., vials) and
the third, if present, is the fixed dose. The entire fixed dose is
then administered to the subject in need thereof.
[0171] In some embodiments, the two or more pharmaceutical
compositions (e.g., vials) in a kit can be administered separately.
For example, a first pharmaceutical composition comprising a first
portion of a fixed dose of a clotting factor is first administered
to a subject in need thereof, and a second pharmaceutical
composition comprising a second portion of a fixed dose of a
clotting factor is then administered to the subject.
[0172] The present invention also identifies the fixed dose that
can treat or prevent one or more bleeding episodes in a subject
regardless of the body weight. Administration of the appropriate
dosing amount for the dosing interval can achieve a plasma trough
level of a clotting activity at least about 1 IU/dl or above 1
IU/dl during the interval in a subject administered with a clotting
factor. In one embodiment, the invention includes a dosing amount
(or ranges of the dosing amount) and a dosing interval (or ranges
of the dosing interval) that can maintain a plasma trough level of
a clotting activity at least about 1 IU/dl (1%) or above 1 IU/dl
(1%), at least about 2 IU/dl (2%) or above 2 IU/dl (2%), at least
about 3 IU/dl (3%) or above 3 IU/dl (3%), at least about 4 IU/dl
(4%) or above 4 IU/dl (4%), at least about 5 IU/dl (5%) or above 5
IU/dl (5%), at least about 6 IU/dl (6%) or above 6 IU/dl (6%), at
least about 7 IU/dl (7%) or above 7 IU/dl (7%), at least about 8
IU/dl (8%) or above 8 IU/dl (8%), at least about 9 IU/dl (9%) or
above 9 IU/dl (9%), at least about 10 IU/dl (10%) or above 10 IU/dl
(10%), at least about 11 IU/dl (11%) or above 11 IU/dl (11%), at
least about 12 IU/dl (12%) or above 12 IU/dl (12%), at least about
13 IU/dl (13%) or above 13 IU/dl (13%), at least about 14 IU/dl
(14%) or above 14 IU/dl (14%), at least about 15 IU/dl (15%) or
above 15 IU/dl (15%), at least about 16 IU/dl (16%) or above 6
IU/dl (16%), at least about 17 IU/dl (17%) or above 17 IU/dl (17%)
at least about 18 IU/dl (8%) or above 18 IU/dl (18%), at least
about 19 IU/dl (19%) or above 19 IU/dl (19%), at least about 20
IU/dl (20%) or above 20 IU/dl (20%) throughout the interval.
[0173] In another embodiments, a plasma trough level of a clotting
factor is maintained between about 1% and about 5%, between about
1% and about 6%, between about 1% and about 7%, between about 1%
and about 8%, between about 1% and about 9%, between about 1% and
about 10%, between about 1% and about 12%, between about 1% and
about 14%, between about 1% and about 15%, between about 1% and
about 17%, between about 1% and about 19%, between about 1% and
about 20%, between about 1% and about 22%, between about 1% and
about 24%, between about 1% and about 25%, between about 1% and
about 30%, between about 1% and about 35%.
[0174] In other embodiments, the trough is 1-5 or 1-3 IU/dl after
about 6, about 7, about 8, about 9, about 10, about 11, about 12,
about 13 or about 14 days after administration of a clotting
factor. In some embodiments, the plasma level of the clotting
factor reaches an average trough of at least about 1 IU/dl after at
least about 6 days or reaches a trough of at least about 1, 2, 3,
4, or 5 IU/dl after at least about 6 days in a subject. In some
embodiments, the plasma level of the clotting factor reaches an
average trough of about 1-5 or 1-3 IU/dl. Such trough or average
trough may be reached after about 6, about 7, about 8, about 9,
about 10, about 11, about 12, about 13, about 14, about 15, about
16, about 17, about 18, about 19, about 20, about 21, about 22,
about 23, about 24, about 25, about 26, about 27, about 28, about
29, about 30, about 31, about 32, about 33, about 34, about 35,
about 36, about 37, about 38, about 39, or about 40 days.
[0175] In some embodiments, a dosing amount (or ranges of the
dosing amount) and a dosing interval (or ranges of the dosing
interval) are selected to reduce or decrease the frequency of
bleeding or bleeding disorder. In other embodiments, the dosing
amount (or ranges of the dosing amount) and the dosing interval (or
ranges of the dosing interval) of a clotting factor stops on-going,
uncontrollable bleeding or bleeding episodes in a subject
administered with the dosing amount during the dosing interval. In
still other embodiments, the dosing amount (or ranges of the dosing
amount) and the dosing interval (or ranges of the dosing interval)
of a clotting factor prevents spontaneous bleeding or bleeding
episodes in a subject susceptible to such spontaneous bleeding or
bleeding episodes.
[0176] In one aspect, a fixed dose of a FIX polypeptide is about
4,000 IU per dose, 6000 IU or about 8,000 IU per dose. In one
embodiment, a dosing interval is at least about every five days,
about every six days, at least about every seven days, at least
about every eight days, at least about every nine days, at least
about every ten days, at least about every 11 days, at least about
every 12 days, at least about every 13 days, at least about every
14 days, at least about every 15 days, at least about every 16
days, at least about every 17 days, at least about every 18 days,
at least about every 19 days, at least about every 20 days, or at
least about every 21 days. In another embodiment, a fixed dose of a
FIX polypeptide is about 4,000 IU per dose and is administered
weekly, i.e., once per week. In other embodiments, a fixed dose of
a FIX polypeptide is about 8,000 IU per dose and is administered
every 10 days or once every two weeks. In yet other embodiments,
the fixed dose of a long-acting FIX polypeptide is not calculated
by the formula:
Number of factor IX IU required (IU)=Body Weight(kg).times.Desired
Factor IX Increase (% or IU/dL).times.1 IU/kg per IU/dL) (A)
[0177] In certain aspects of the invention, the method, the use,
the pharmaceutical composition, or the kit further comprises
administering an additional dosing amount of a clotting factor.
[0178] In certain embodiments, the fixed dosing strategy is a
stratified dosing regimen. For example, the fixed dose can be
stratified into two or more dose sizes based on specified weight
categories. The weight categories can be low body weight, normal
body weight, and high body weight. In one embodiment, the fixed
dose is stratified into three dose sizes suitable for subjects with
low, normal, or high body weight. The normal, low, or high body
weight can be determined based on age, height, gender, frame size,
general health, or any combination thereof or independently of age,
height, gender, frame size, general health, or any combination
thereof. In another embodiment, a subject has a low body weight,
and the fixed dose of a long-acting FIX polypeptide is about 5,000
IU per dose or about 6,000 IU per dose, which is administered at an
interval longer than 7 days, e.g., every 10 days. In other
embodiments, a subject has a normal body weight and the fixed dose
is about 7500 IU per dose or about 8000 IU per dose, which is
administered at an interval longer than 7 days, e.g., every 10
days. In some embodiments, a subject has a high body weight and the
fixed dose is about 10000 IU per dose administered every 10 days or
about 12000 IU per dose administered every 10 days.
[0179] The dosing interval can, alternatively, be an individualized
interval that is determined for each subject based on
pharmacokinetic data or other information about that subject. The
individualized dose/dosing interval combination may be the same as
those for fixed interval regimens in the preceding paragraphs, or
may differ. The regimen can initially be at a fixed dosing
interval, and then it can change to an individualized dosing
interval.
[0180] In certain embodiments of the invention, the method of the
invention further comprises measuring a baseline FIX activity of a
subject prior to the initial administration of a FIX polypeptide.
Measuring of a baseline FIX activity can employ any known clotting
assays in the art, e.g., one step aPTT assay, two step chromogenic
assay, ROTEM, TGA, or etc.
[0181] In one aspect, a fixed dose of a FVIII polypeptide is about
2000 IU, about 2,500 IU, about 3,000 IU, about 3,500 IU, or about
4,000 IU per dose. In one embodiment, the fixed dose is
administered twice a week (i.e., two times per week). In another
embodiment, the fixed dose is administered weekly (i.e., once a
week). In other embodiments, the entire fixed dose is administered
to the subject, i.e., no portion of the composition is left unused.
In yet other embodiments, the fixed dose of a long-acting FVIII
polypeptide is not calculated by the formula:
Number of factor FVIII IU required (IU)=Body Weight
(kg).times.Desired Factor FVIII Increase (IU/dL or % of
normal).times.0.5(IU/kg per IU/dL) (B)
IV. METHODS OF MAKING
[0182] A clotting factor can be manufactured in a host cell
comprising a vector encoding the clotting factor. As used herein,
an expression vector refers to any nucleic acid construct which
contains the necessary elements for the transcription and
translation of an inserted coding sequence, or in the case of an
RNA viral vector, the necessary elements for replication and
translation, when introduced into an appropriate host cell.
Expression vectors can include plasmids, phagemids, viruses, and
derivatives thereof.
[0183] A gene expression control sequence as used herein is any
regulatory nucleotide sequence, such as a promoter sequence or
promoter-enhancer combination, which facilitates the efficient
transcription and translation of the coding nucleic acid to which
it is operably linked. The gene expression control sequence may,
for example, be a mammalian or viral promoter, such as a
constitutive or inducible promoter. Constitutive mammalian
promoters include, but are not limited to, the promoters for the
following genes: hypoxanthine phosphoribosyl transferase (HPRT),
adenosine deaminase, pyruvate kinase, beta-actin promoter, and
other constitutive promoters. Exemplary viral promoters which
function constitutively in eukaryotic cells include, for example,
promoters from the cytomegalovirus (CMV), simian virus (e.g.,
SV40), papilloma virus, adenovirus, human immunodeficiency virus
(HIV), Rous sarcoma virus, cytomegalovirus, the long terminal
repeats (LTR) of Moloney leukemia virus, and other retroviruses,
and the thymidine kinase promoter of herpes simplex virus. Other
constitutive promoters are known to those of ordinary skill in the
art. The promoters useful as gene expression sequences of the
invention also include inducible promoters. Inducible promoters are
expressed in the presence of an inducing agent. For example, the
metallothionein promoter is induced to promote transcription and
translation in the presence of certain metal ions. Other inducible
promoters are known to those of ordinary skill in the art.
[0184] Examples of vectors include, but are not limited to viral
vectors or plasmid vectors. Plasmid vectors have been extensively
described in the art and are well-known to those of skill in the
art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
In the last few years, plasmid vectors have been found to be
particularly advantageous for delivering genes to cells in vivo
because of their inability to replicate within and integrate into a
host genome. These plasmids, however, having a promoter compatible
with the host cell, can express a peptide from a gene operably
encoded within the plasmid. Some commonly used plasmids available
from commercial suppliers include pBR322, pUC18, pUC19, various
pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and
pBlueScript. Additional examples of specific plasmids include
pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number
V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1,
catalog number V53220, all from Invitrogen (Carlsbad, Calif.).
Other plasmids are well-known to those of ordinary skill in the
art. Additionally, plasmids may be custom designed using standard
molecular biology techniques to remove and/or add specific
fragments of DNA.
[0185] The expression vector or vectors are then transfected or
co-transfected into a suitable target cell, which will express the
polypeptides. Transfection techniques known in the art include, but
are not limited to, calcium phosphate precipitation (Wigler et al.
(1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J
1:841), and liposome-based reagents. A variety of host-expression
vector systems may be utilized to express the proteins described
herein including both prokaryotic and eukaryotic cells. These
include, but are not limited to, microorganisms such as bacteria
(e.g., E. coli) transformed with recombinant bacteriophage DNA or
plasmid DNA expression vectors containing an appropriate coding
sequence; yeast or filamentous fungi transformed with recombinant
yeast or fungi expression vectors containing an appropriate coding
sequence; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing an appropriate
coding sequence; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus or tobacco
mosaic virus) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing an appropriate coding
sequence; or animal cell systems, including mammalian cells (e.g.,
HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).
[0186] In one embodiment, the host cell is a eukaryotic cell. As
used herein, a eukaryotic cell refers to any animal or plant cell
having a definitive nucleus. Eukaryotic cells of animals include
cells of vertebrates, e.g., mammals, and cells of invertebrates,
e.g., insects. Eukaryotic cells of plants specifically can include,
without limitation, yeast cells. A eukaryotic cell is distinct from
a prokaryotic cell, e.g., bacteria.
[0187] In certain embodiments, the eukaryotic cell is a mammalian
cell. A mammalian cell is any cell derived from a mammal. Mammalian
cells specifically include, but are not limited to, mammalian cell
lines. In one embodiment, the mammalian cell is a human cell. In
another embodiment, the mammalian cell is a HEK 293 cell, which is
a human embryonic kidney cell line. HEK 293 cells are available as
CRL-1533 from American Type Culture Collection, Manassas, Va., and
as 293-H cells, Catalog No. 11631-017 or 293-F cells, Catalog No.
11625-019 from Invitrogen (Carlsbad, Calif.). In some embodiments,
the mammalian cell is a PER.C6.RTM. cell, which is a human cell
line derived from retina. PER.C6.RTM. cells are available from
Crucell (Leiden, The Netherlands). In other embodiments, the
mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are
available from American Type Culture Collection, Manassas, Va.
(e.g., CHO-K1; CCL-61). In still other embodiments, the mammalian
cell is a baby hamster kidney (BHK) cell. BHK cells are available
from American Type Culture Collection, Manassas, Va. (e.g.,
CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell,
which is a hybrid cell line of a HEK293 cell and a human B cell
line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).
[0188] The method can further comprise purification steps. Various
known purifications steps are well known in the art.
[0189] Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention. All
patents and publications referred to herein are expressly
incorporated by reference.
EXAMPLES
Example 1
Population Pharmacokinetic Analysis of a Long-Acting Recombinant
Factor IX-Fc Fusion Protein (rFIXFc) in Patients with Severe
Hemophilia B
[0190] BACKGROUND: Population pharmacokinetic (popPK) models are
developed to understand the sources of variability in dose
requirements (covariates) and to help individualize dosing regimens
if necessary. Dosing histories and patient-specific data are used
to gain an understanding of drug disposition in order to discern
specific demographic and/or clinical factors that may be predictors
of PK parameters. By characterizing the population PK of
long-acting FIX-Fc (rFIXFc) in patients with severe hemophilia B
(.ltoreq.2 IU/dL plasma FIX activity), a model of estimated
population PK parameters of rFIXFc can be established. This model
may assist physicians who wish to tailor dosing for individual
patients with sparse PK samples. This model may also help determine
the suitability of rFIXFc for a fixed dosing regimen.
[0191] METHODS: Male subjects with severe hemophilia B were
included from a phase 1/2a clinical study (n=12) and a phase 3
clinical study (B-LONG, n=123) of rFIXFc. Male subjects with severe
hemophilia B were treated with long-lasting recombinant FIX-Fc
(rFIXFc) in an amount of 50 IU/kg or 100 IU/kg. The subjects ranged
in age from 12 to 76 years and in body weight from 45 to 186 kg.
The modeling dataset included 135 baseline PK profiles at Week 1,
as well as 21 repeat PK profiles at Week 26, with a total of 1400
measured FIX activity records. The final population PK model was
validated using 1027 trough/peak FIX activity records from 119
patients.
[0192] In the popPK analysis, plasma FIX activity was measured by
the one-stage (activated partial thromboplastin time) clotting
assay. Corrected FIX activity was calculated using the formula:
Corrected FIX activity=Measured FIX activity -Baseline-Residual
decay.
[0193] Baseline FIX activity was defined as the lowest level of
activity (LLACT) recorded at screening, predose, postdose, or from
historical clinical records. The baseline is defined as 0 when the
LLACT is less than 1% (lower limit of quantification). The baseline
FIX activity is equal to LLACT when LLACT is from 1% to 2% (i.e.,
1.ltoreq.LLACT.ltoreq.2).
[0194] Prestudy residual decay was performed using terminal
half-life obtained from a noncompartmental analysis of the
individual data by the following formula:
Residual decay=(predose-baseline).times.e.sup.-decay
rate.times.time.
[0195] Clearance was presented by the following formula:
CL=Typical CL.times.(BW/Typical BW).sup.exponent, where typical BW
is 73 kg.
[0196] Volume of distribution was presented by the following
formula:
V=Typical V.times.(BW/Typical BW).sup.exponent, where typical BW is
73 kg.
[0197] For the popPK model development, NONMEM VII version 1.0
(ICON Development Solutions, Ellicott City, Md.) was used. The
modeling and qualification steps are presented below in Table
1.
TABLE-US-00001 TABLE 1 Modeling and Qualification Steps Steps Model
selection Base model and Inter-individual Base Model, IIV on
CL/V1/Q2/V2/Q3 variability (IIV) evaluation Inter-occasion
variability (IOV) Base Model with IOV on CL and V1 evaluation
Covariate Modelling Final model, body weight as covariate on CL and
V1 Internal qualification (bootstrap and VPC) External
qualification using trough/peak records CL, clearance; V, volume of
distribution; Q, inter-compartmental clearance; VPC, visual
predictive check
[0198] A first order conditional estimation with interaction method
(FOCET) was used to estimate the popPK parameters. Residual errors
were modeled as combined proportional and additive errors. Stepwise
forward addition (p<0.005) and backward elimination (p<0.001)
covariate modeling was performed. Potential covariates assessed in
this analysis included: body weight (BW), Age, Race, Blood type,
Human Immunodeficiency Virus status, Hepatitis C Virus status,
haematocrit, IgG.sub.1 and albumin concentration, and FIX
genotype.
[0199] Model qualifications included bootstrap, visual predictive
check (VPC) and validation with trough/peak records. The mean
relative prediction error (an indicator of accuracy) was calculated
as:
1 N i = 1 i = N [ DV - IPRED ] DV ##EQU00001##
[0200] RESULTS: The rFIXFc disposition was best described by a
three-compartment base model (FIG. 1). The model was further
improved by including intra-subject random changes at different
occasions (i.e., inter-occasion variability, IOV) for CL and V1
(FIG. 2). IOV was smaller than inter-individual variability (IIV),
indicating that individual PK was more accurate than the mean popPK
for individual PK prediction.
[0201] BW was the only statistically significant covariate on CL
and V1 (volume of the central compartment). However, the impact of
body weight on the PK of rFIXFc was limited. Body weight was found
to be a significant covariate for rFIXFc disposition (FIG. 3),
although the impact of BW was limited. For example, the BW exponent
on CL and V1 was 0.436 and 0.396, respectively, and inclusion of BW
reduced inter-individual variability (IIV) for both CL and V1 only
by 3.4% and 2.5%, respectively. None of the other covariates
assessed, including age, race, blood type or genotype, were
significant covariates in this model.
[0202] The final popPK model is summarized below in Table 2.
TABLE-US-00002 TABLE 2 Summary of the final rFIXFc population
pharmacokinetic model. Population 95% non-parametic CI IIV.sup.b
IOV Parameter Estimate from bootstrap.sup.a (%) (%) CL = Typical CL
.times. ( BW 73 ) 0.436 ##EQU00002## Typical CL for a 73 kg 2.39
2.29, 2.49 17.7 15.1 subject (dL/h) BW exponent on CL 0.436 0.272,
0.584 V1 = Typical V1 .times. ( BW 73 ) 0.396 ##EQU00003## Typical
V1 for a 73 kg 71.4 58.5, 76.0 21.7 17.4 subject (dL) BW exponent
on V1 0.396 0.169, 0.580 Q2 (dL/h) 1.67 1.35, 1.89 35.8 -- V2 (dL)
87.0 79.0, 95.5 46.2 -- Q3 (dL/h) 39.3 16.6, 141 -- -- V3 (dL) 39.9
36.6, 52.4 37.7 -- Residual Error: Proportional 10.6% Additive 0.24
IU/dL CI, confidence interval; IIV, inter-individual variability;
IOV, inter occasion variability; CL, clearance; BW, body weight; V,
volume of distribution; Q, inter-compartmental clearance
[0203] For a typical 73 kg subject, the predicted popPK values for
clearance, volume of central compartment, and volume of
distribution at steady state are 2.39 dL/h, 71.4 dL, and 198 dL,
respectively. Goodness-of-fit plots show that the predicted popPK
data generated by the model closely mimic the observed FIX activity
data (FIG. 4).
[0204] The results of the popPK model were validated using the
observed FIX activity data. The median and 80% interval for
observed and predicted FIX activity time profiles nearly
overlapped, indicating that the final model was able to reproduce
both the central tendency and variability of the observed FIX
activity data on the time scale (FIG. 5). The strong correlation
between observed and predicted FIX activities in the trough/peak
dataset suggested that the final popPK model is predictive (FIG.
6).
[0205] Finally, the overall relative prediction error was -3.23%
with a 95% confidence interval of -5.27% to -1.23%. Post hoc
estimates from this popPK analysis were very similar to the results
from the conventional PK analysis shown below in Table 3.
TABLE-US-00003 TABLE 3 Post hoc empirical Bayesian estimates of key
PK parameters. Phase 3 Phase 1/2a Parameter Mean (SD) Mean (SD)
Clearance (CL), mL/h/kg 3.42 (0.89) 2.82 (0.58) Volume of central
compartment (V1), mL 102 (29.6) 96.2 (24.7) Incremental in vivo
recovery, IU/dL per 1.02 (0.45) 1.04 (0.19) IU/kg Volume of
distribution at steady-state 297 (90.5) 234 (70.8) (Vss), mL/kg
Terminal Half-life, h 86.7 (27.9) 70.9 (13.9) Mean residence time
(MRT), h 89.4 (25.9) 82.5 (15.5) SD, standard deviation
[0206] CONCLUSIONS: The three-compartment popPK model predicted
disposition of rFIXFc with modest inter-individual variability
(IIV). Individual PK parameters derived from the three-compartment
popPK model were similar to those derived from the two-compartment
conventional PK analysis, indicating a limited 3rd compartment
contribution. For a typical 73 kg subject, the popPK model
predicted a clearance of 2.39 dL/h; volume of central compartment
of 71.4 dL; and volume of distribution at steady state of 198 dL.
The only significant covariate assessed in the popPK model was BW,
although its impact on rFIXFc PK variability was limited.
[0207] Drugs with body weight effect on clearance
(.theta..sub.BW.sub._.sub.CL) and body weight effect on the central
volume of distribution (.theta..sub.BW.sub._.sub.V1) equal to or
less than 0.5 are considered good candidates for fixed dosing
regimens due to the limited impact of patient body weight on PK
variability. Here, rFIXFc had .theta..sub.BW.sub._.sub.CL and
.theta..sub.BW.sub._.sub.V1 values of 0.436 and 0.396, respectively
(Table 2). Furthermore, the inclusion of BW in the population PK
model resulted in a modest reduction of approximately 3% in IIV for
both CL and V1. These results indicate that body weight had a
minimal impact on PK variability and suggest that rFIXFc is
suitable for a fixed dosing regimen.
[0208] The final popPK model can be used to simulate dosing
regimens and intervals for routine prophylaxis, control and
prevention of bleeding episodes, and peri-operative management.
This model may assist physicians who wish to tailor dosing for
individual patients with sparse PK samples.
Example 2
Fixed Dosing
[0209] The body weight of adult patients has a limited impact on PK
variability among patients. Therefore, rFIXFc is suitable for fixed
dosing regimens that do not use the formula:
Number of factor FIX IU required (IU)=Body Weight
(kg).times.Desired Factor FIX Increase (IU/dL or % of
normal).times.0.5(IU/kg per IU/dL).
[0210] In this example, fixed dose regimens are established using
vials of rFIXFc that contain 2,000 IU per vial. In one alternative,
the entire population of adult patients is treated with 2 vials of
rFIXFc once weekly. Alternatively, stratified fixed dosing is
applied based on the BW range in which the patient belongs to.
[0211] METHODS: Patients with hemophilia B are categorized into one
of three categories: (i) low body weight; (ii) normal body weight;
and (iii) high body weight. Patients weighing less than 57 kg are
categorized as low body weight. Patients weighing between 57 and
104 kg are categorized as normal body weight. Patients weighing
more than 104 kg are categorized as high body weight.
[0212] Patients in the low body weight category are treated with a
single vial of fixed dose long acting FIXFc (i.e., 2,000 IU total)
once weekly. Patients in the normal body weight category are
treated with two vials of fixed dose long-acting rFIXFc (i.e.,
4,000 IU total) once weekly. Patients in the high body weight
category are treated with three vials of fixed dose long-acting
rFIXFc (i.e., 6,000 IU total) once weekly.
[0213] RESULTS: The PK properties of long-acting rFIXFc are
minimally affected by the BW. As FIGS. 7A and 7B show, the
97.5.sup.th, median, and 2.5.sup.th percentiles of the simulated
FIX activity-time profiles at steady state in 1000 subjects
following fixed dosing (4000 IU once weekly and 8000 IU every 10
days; dotted lines) significantly overlap with those of BW-based
dosing (50 IU/kg once weekly and 100 IU/kg every 10 days; solid
lines). FIG. 8 shows that the percentages of population within the
target therapeutic range following the fixed dosing and body weight
(BW)-based dosing approaches in the BW-stratified population are
similar. These data demonstrate that the clotting factors having a
wide therapeutic window can be used for fixed dosing regimen: for
example, this allows the physician to treat patients with fixed
dose regimens of long-acting rFIXFc, eliminating the need to use
formulaic dose calculation methods.
Example 3
Population Pharmacokinetic Analysis of a Long-Acting Recombinant
Factor VIII-Fc Fusion Protein (rFVIIIFc) in Patients with Severe
Hemophilia A
[0214] By characterizing the population PK of long-acting FVIII-Fc
(rFVIIIFc) in patients with severe hemophilia A, a model of
estimated population PK parameters of rFVIIIFc can be established.
This model may assist physicians who wish to tailor dosing for
individual patients with sparse PK samples. This model may also
help determine the suitability of rFVIIIFc for a fixed dosing
regimen.
[0215] Objectives: To characterize the activity-time profiles of
rFVIIIFc in hemophilia A patients by population PK analysis and to
identify intrinsic covariates that may affect the variability of
rFVIIIFc PK.
[0216] The modeling dataset included activity-time profiles of 180
subjects (15 from a Phase 1/2a study and 165 from a Phase 3 study
[A-LONG], collected over .ltoreq.52 weeks of treatment). Subjects
were 12 to 65 years old and weighed 41-132 kg. The analysis was
done with NONMEM 7 software, and included model building, covariate
search, and model qualification steps.
[0217] A 2-compartmental model adequately described the activity of
rFVIIIFc. The population estimate for clearance (CL)=1.65 dL/h;
volume of distribution at steady state (Vss)=44.4 dL. The
inter-individual variability (IIV) of CL was moderate (24.3%) and
central volume of distribution (V1) was low (13.4%). The
inter-occasional variability (IOV) of both CL and V1 was low (20.6
and 12.0% respectively). The additive residual error was very low
(0.208 IU/dL), as was the proportional residual error (13.6%),
approximating the variability of the one-stage clotting assay for
FVIII activity. Von Willebrand Factor (VWF) level was identified as
the major covariate for CL; higher levels of VWF yielded lower
clearance values, reflecting the protective role that VWF has on
FVIII activity. Body Weight (BW) and Haematocrit (HCT) were
identified as weak covariates on V1.
[0218] This is the first population PK analysis that systematically
describes and characterizes the prolonged activity profile of
long-acting rFVIIIFc. The population PK model of rFVIII activity
adequately describes the observed activity-time profiles. The
clearance of rFVIIIFc activity is lower than the clearance observed
for ADVATE.RTM., resulting in longer duration of activity. The low
IIV underlines the consistency and homogeneity of the activity
profiles. The low IOV indicates that rFVIIIFc maintains stable and
predictable activity with long term administration over time. The
set of covariates identified is physiologically relevant.
Therefore, the population model developed can be used to simulate
various dosing scenarios in support of dosing regimen selection and
other decision making related to rFVIIIFc therapy. This approach
represents an advance over the current utilitarian approach,
wherein a regimen is not determined to be ineffective until after a
patient has a bleeding episode.
[0219] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of the present invention. Therefore, such
adaptations and modifications are intended to be within the meaning
and range of equivalents of the disclosed embodiments, based on the
teaching and guidance presented herein. It is to be understood that
the phraseology or terminology herein is for the purpose of
description and not of limitation, such that the terminology or
phraseology of the present specification is to be interpreted by
the skilled artisan in light of the teachings and guidance.
[0220] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
[0221] All patents and publications cited herein are incorporated
by reference herein in their entirety.
TABLES
TABLE-US-00004 [0222] TABLE 4 Polynucleotide Sequences: FIX-Fc A.
FIX-Fc Chain DNA Sequence (SEQ ID NO: 1, which encodes SEQ ID NO:
2) pSYN-FIX-030 Nucleotide sequence (nt 1 to 7583): FIX exon 1
(signal peptide, 1st amino acid propeptide): nt 690-777 FIX mini
intron: nt 778-1076 FIX propeptide sequence: nt 1077-1126 Mature
FIX sequence: nt 1127-2371 Fc: nt 2372-3052
gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatat-
a
tggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacg-
t
caataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacgg-
t
aaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaa-
t
ggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtc-
a
tcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggattt-
c
caagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgt-
a
acaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctgg-
c
taactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttcgcga-
c
gtacggccgccaccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgcctttta-
g
gatatctactcagtgctgaatgtacaggtttgtttccttttttaaaatacattgagtatgcttgccttttagat-
a
tagaaatatctgatgctgtcttcttcactaaattttgattacatgatttgacagcaatattgaagagtctaaca-
g
ccagcacgcaggttggtaagtactgtgggaacatcacagattttggctccatgccctaaagagaaattggcttt-
c
agattatttggattaaaaacaaagactttcttaagagatgtaaaattttcatgatgttttcttttttgctaaaa-
c
taaagaattattcttttacatttcagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaaga-
g
gtataattcaggtaaattggaagagtttgttcaagggaatctagagagagaatgtatggaagaaaagtgtagtt-
t
tgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatc-
a
gtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccct-
t
tggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgta-
a
aaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtg-
a
accagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgagactgttt-
t
tcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcat-
t
taatgacttcactcgggttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatg-
g
taaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaa-
c
tggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatg-
t
gattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaac-
t
ggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcc-
t
caaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagt-
a
ccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttct-
g
tgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaag-
g
gaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatatacca-
a
ggtgtcccggtatgtcaactggattaaggaaaaaacaaagctcactgacaaaactcacacatgcccaccgtgcc-
c
agctccggaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctccc-
g
gacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtgg-
a
cggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcg-
t
cctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag-
c
ccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc-
g
ggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtgg-
a
gtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttct-
t
cctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatg-
a
ggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattcagacatgataaga-
t
acattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgct-
a
ttgctttatttgtaaccattataagctgcaataaacaagttggggtgggcgaagaactccagcatgagatcccc-
g
cgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcgqtgga-
a
tcgaaatctcgtagcacgtgtcagtcctgctcctcggccacgaagtgcacgcagttgccggccgggtcgcgcag-
g
gcgaactcccgcccccacggctgctcgccgatctcggtcatggccggcccggaggcgtcccggaagttcgtgga-
c
acgacctccgaccactcggcgtacagctcgtccaggccgcgcacccacacccaggccagggtgttgtccggcac-
c
acctggtcctggaccgcgctgatgaacagggtcacgtcgtcccggaccacaccggcgaagtcgtcctccacgaa-
g
tcccgggagaacccgagccggtcggtccagaactcgaccgctccggcgacgtcgcgcgcggtgagcaccggaac-
g
gcactggtcaacttggccatggtttagttcctcaccttgtcgtattatactatgccgatatactatgccgatga-
t
taattgtcaacacgtgctgatcagatccgaaaatggatatacaagctcccgggagctttttgcaaaagcctagg-
c
ctccaaaaaagcctcctcactacttctggaatagctcagaggcagaggcggcctcggcctctgcataaataaaa-
a
aaattagtcagccatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggc-
g
ggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttcca-
c
acctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacac-
c
ctcgtcgagctagcttcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaa-
g
ttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgt-
g
tactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttt-
t
cgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggtta-
t
ggcccttgcgtgccttgaattacttccacctggctccagtacgtgattcttgatcccgagctggagccaggggc-
g
ggccttgcgctttaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtg-
c
gaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacct-
g
ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaggatctgcacactggtatttcggttttt-
g
gggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcca-
c
cgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc-
c
cgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgct-
c
cagggggctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaggggcc-
t
ttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctgg-
a
gcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtg-
g
agactgaagttaggccagcttggcacttgatgtaattctccttggaacttgccctttttgagtttggatcttgg-
t
tcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaacacgtggtcgcggc-
c
gcgccgccaccatggagacagacacactcctgctatgggtactgctgctccgggttccaggttccactggtgac-
a
aaactcacacatgcccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaa-
c
ccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccct-
g
aggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtac-
a
acagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgc-
a
aggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacca-
c
aggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacccgcctggtcaaaggc-
t
tctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctccc-
g
tgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaac-
g
tcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt-
a
aatgactcgagagatctggccggctgggcccgtttcgaaggtaagcctatccctaaccctctcctcggtctcga-
t
tctacgcgtaccggCcatcaccaccatcaccattgagtttaaacccgctgatcagcctcgactgtgccttctag-
t
tgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttc-
c
taataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagga-
c
agcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcgga-
a
agaaccagtggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcc-
a
gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc-
a
caaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctagaa-
g
ctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcg-
t
ggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgc-
a
cgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacg-
a
cttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttct-
t
gaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacct-
t
cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagc-
a
gcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtgga-
a
cgaaaactcacgttaagggattttggtcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc-
t
cgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaag-
c
ggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatg-
c
ggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaa-
a
taccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgct-
a ttacgcca B. Fc DNA sequence (mouse Ig.kappa. signal peptide
underlined) (SEQ ID NO: 3, which encodes SEQ ID NO: 4) This is the
Fc cassette from pSYN-FIX-030. In addidtion, there is a separate Fc
expression cassette that was transfected into the cell line in
plasmid pSYN-Fc-015 that encodes the same amino acid sequence, but
contains a few noncoding changes. The second copy of Fc encoding
sequence enables a better monomer: dimer ratio.
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacaaaactcacac-
a
tgcccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacac-
c
ctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagtt-
c
aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgta-
c
cgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaa-
c
aaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacac-
c
ctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccag-
c
gacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactc-
c
gacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatg-
c
tccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa
TABLE-US-00005 TABLE 5 Polypeptide Sequences: FIX-Fc FIX-Fc Monomer
Hybrid: created by coexpressing FIX-Fc and Fc chains. A. FIX-Fc
chain (SEQ ID NO: 2): (28 amino acid signal sequence underlined, 18
amino acid propeptide double underlined, Fc portion in italics.)
The C-terminal lysine is not present in either subunit; this
processing is often observed in recombinant proteins produced in
mammalian cell culture, as well as with plasma derived proteins.
FIXFC-SC SUBUNIT: FIX SignalPeptide: -46 MQRVNMIMAE SPGLITICLL
GYLLSAEC FIX Propeptide: -18 TVFLDHENAN KILNUKR 1 YNSGKLEEFV
QGNLERECME EKCSFEEARE VFENTERTTE FWKQYVDGDQ 51 CESNPCLNGG
SCKDDINSYE CWCPFGFEGK NCELDVTCNI KNGRCEQFCK 101 NSADNKVVCS
CTEGYRLAEN QKSCEPAVPF PCGRVSVSQT SKLTRAETVF 151 PDVDYVNSTE
AETILDNITQ STQSFNDFTR VVGGEDAKPG QFPWQVVLNG 201 KVDAFCGGSI
VNEKWIVTAA HCVETGVKIT VVAGEHNIEE TEHTEQKRNV 251 IRIIPHHNYN
AAINKYNHDI ALLELDEPLV LNSYVTPICI ADKEYTNIFL 301 KFGSGYVSGW
GRVFRRGR6A LVLQYLRVPL VDRATCLRST KFTIYNNMFC 351 AGFHEGGRDS
CQGDSGGPHV TEVEGTSFLT GIISWGEECA MKGKYGIYTK 401 VSRYVNWIKE
KTKLTDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR 451 TPEVTCVVVD
VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV 501 LTV1HQDWIN
GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 551 LELTENQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF 601 LYSKLTVDKS
RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK B. Fc chain (SEQ ID NO: 4) 20
amino acid heterologous mouse Ig.kappa. light chain signal peptide
(underlined): -20 METDTLLLWV LLLWVPGSTG Mature Fc sequence
(corresponding to human IgG1 amino acids 221 to 447, EU numbering)
1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 51
PEVKFNWYVD GVEVENAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 101
CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK 151
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG 201
NVFSCSVMHE ALHNHYTQKS LSLSPGK
TABLE-US-00006 TABLE 6 Polynucleotide Sequence: FVIII-Fc A.
B-Domain Deleted FVIIIFc (i) B-Domain Deleted FVIIIFc Chain DNA
Sequence (FVIII signal peptide underlined. Fc region in bold) (SEQ
ID NO: 5, which encodes SEQ ID NO: 6) 661 A TGCAAATAGA GCTCTCCACC
TGCTTCTTTC 721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA
CTACCTGGGT GCAGTGGAAC 781 TGTCATGGGA CTATATGCAA AGTGATCTCG
GTGAGCTGCC TGTGGACGCA AGATTTCCTC 841 CTAGAGTGCC AAAATCTTTT
CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG 901 TAGAATTCAC
GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC 961
TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG
1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT
TCTGAGGGAG 1081 CTGAATATGT TGATCAGACC AGTCAAAGGG AGAAAGAAGA
TGATAAAGTC TTCCCTGGTG 1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA
AAGAGAATGG TCCAATGGCC TCTGACCCAC 1201 TGTGCCTTAC CTACTCATAT
CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261 TCATTGGAGC
CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321
TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA
1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG
CCTAAAATGC 1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT
GATTGGATGC CACAGGAAAT 1501 CAGTCTATTG GCATGTGATT GGAATGGGCA
CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561 AAGGTCACAC ATTTCTTGTG
AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621 CTTTCCTTAC
TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681
TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG
1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA CTATGATGAT
GATCTTACTG 1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC
TCCTTCCTTT ATCCAAATTC 1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT
GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921 ACTGGGACTA TGCTCCCTTA
GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981 TGAACAATGG
CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041
CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT
2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA
GCAAGCAGAC 2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC
TTTGTATTCA AGGAGATTAC 2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC
CAATTCTGCC AGGAGAAATA TTCAAATATA 2281 AATGGACAGT GACTGTAGAA
GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341 ATTACTCTAG
TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401
TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA
2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA
GAGAATATAC 2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA
TCCAGAGTTC CAAGCCTCCA 2581 ACATCATGCA CAGCATCAAT GGCTATGTTT
TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641 ATGAGGTGGC ATACTGGTAC
ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701 TCTTCTCTGG
ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761
CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT
2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT
TCTAGTTGTG 2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA
TATTTCAGCA TACTTGCTGA 2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT
TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001 ATCAACGGGA AATAACTCGT
ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061 ATACCATATC
AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121
AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC
3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT
CAGAGTGGCA 3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC
TGATGGCTCC TTTACTCAGC 3301 CCTTATACCG TGGAGAACTA AATGAACATT
TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361 AAGTTGAAGA TAATATCATG
GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421 ATTCTAGCCT
TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT 3481
TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA
3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC
CTGGAAAAAG 3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA
CACTAACACA CTGAACCCTG 3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT
TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721 CCAAAAGCTG GTACTTCACT
GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781 AGATGGAAGA
TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA 3841
TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA
3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG
TTCACTGTAC 3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA
TCCAGGTGTT TTTGAGACAG 4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT
GGCGGGTGGA ATGCCTTATT GGCGAGCATC 4081 TACATGCTGG GATGAGCACA
CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141 GAATGGCTTC
TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201
GGGCCCCAAA GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG AGCACCAAGG
4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC
GGCATCAAGA 4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC
TCAGTTTATC ATCATGTATA 4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC
GAGGAAATTC CACTGGAACC TTAATGGTCT 4441 TCTTTGGCAA TGTGGATTCA
TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501 CTCGATACAT
CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561
TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT
4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC
TGGTCTCCTT 4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG
GAGACCTCAG GTGAATAATC 4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA
AGACAATGAA AGTCACAGGA GTAACTACTC 4801 AGGGAGTAAA ATCTCTGCTT
ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861 AAGATGGCCA
TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA 4921
ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC
4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG
GTTCTGGGCT 5041 GCGAGGCACA GGACCTCTAC GACAAAACTC ACACATGCCC
ACCGTGCCCA GCTCCAGAAC 5101 TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC
CCCCAAAACC CAAGGACACC CTCATGATCT 5161 CCCGGACCCC TGAGGTCACA
TGCCTGGTGG TGGACCTGAG CCACGAAGAC CCTGAGGTCA 5221 AGTTCAACTG
GTACGTGGAC GGCGTGGAGG TCCATAATGC CAAGACAAAG CCGCGGGAGG 5281
AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CACCACTGGC
5341 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC
CCCATCGAGA 5401 AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA
GGTGTACACC CTGCCCCCAT 5461 CCCGGGATGA GCTGACCAAG AACCAGGTCA
GCCTGACCTG CCTGGTCAAA GGCTTCTATC 5521 CCAGCGACAT CGCCGTGGAG
TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA 5581 CGCCTCCCGT
GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA 5641
AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA
5701 ACCACTACAC GCAGAAGACC CTCTCCCTGT CTCCGGGTAA A (ii) Fc DNA
sequence mouse Ig.kappa. signal peptide underlined) (SEQ ID NO: 3,
which encodes SEQ ID NO: 4) 7981 ATGGA GACAGACACA 8041 CTCCTGCTAT
GGGTACTGCT GCTCTGGGTT CCAGGTTCCA CTGGTGACAA AACTCACACA 8101
TGCCCACCGT GCCCAGCACC TGAACTCCTG GGAGGACCGT CAGTCTTCCT CTTCCCCCCA
8161 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT
GGTGGTGGAC 8221 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG
TGGACGGCGT GGAGGTGCAT 8281 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG
TACAACAGCA CGTACCGTGT GGTCAGCGTC 8341 CTCACCGTCC TGCACCAGGA
CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 8401 AAAGCCCTCC
CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 8461
CCACAGGTGT ACACCCTGCC CCCATCCCGC GATGAGCTGA CCAAGAACCA GGTCAGCCTG
8521 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA
GAGCAATGGG 8581 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG
ACTCCGACGG CTCCTTCTTC 8641 CTCTACAGCA AGCTCACCGT GGACAAGAGC
AGGTGGCAGC AGGGGAACGT CTTCTCATGC 8701 TCCGTGATGC ATGAGGCTCT
GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 8761 GGTAAA B.
Full-length FVIIIFc (i) Full-length FVIIIFc DNA Sequence (FVIII
signal peptide underlined, Fc region in bold) (SEQ ID NO: 7, which
encodes SEQ ID NO: 8) 661 ATG CAAATAGAGC TCTCCACCTG 721 CTTCTTTCTG
TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC 781
AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG
841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT
ACAAAAAGAC 901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT
AAGCCAAGGC CACCCTGGAT 961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA
GGTTTATGAT ACAGTGGTCA TTACACTTAA 1021 GAACATGGCT TCCCATCCTG
TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC 1081 TGAGGGAGCT
GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT 1141
CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC
1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA
AAGACTTGAA 1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG
AGTCTGGCCA AGGAAAAGAC 1321 ACAGACCTTG CACAAATTTA TACTACTTTT
TGCTGTATTT GATGAAGGGA AAAGTTGGCA 1381 CTCAGAAACA AAGAACTCCT
TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC 1441 TAAAATGCAC
ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA 1501
CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT
1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT
TGGAAATCTC 1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC
CTTGGACAGT TTCTACTGTT 1681 TTGTCATATC TCTTCCCACC AACATGATGG
CATGGAAGCT TATGTCAAAG TAGACAGCTG 1741 TCCAGAGGAA CCCCAACTAC
GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATGA
1801 TCTTACTGAT TCTGAAATGG ATGTGGTCAG GTTTGATGAT GACAACTCTC
CTTCCTTTAT 1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG
GTACATTACA TTGCTGCTGA 1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT
CCTCGCCCCC GATGACAGAA GTTATAAAAG 1981 TCAATATTTG AACAATGGCC
CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT 2041 GGCATACACA
GATGAAACCT TTAAGACTCG TGAAGCTATT CAGCATGAAT CAGGAATCTT 2101
GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA AGAATCAAGC
2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT
TGTATTCAAG 2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA
ATTCTGCCAG GAGAAATATT 2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA
TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341 GACCCGCTAT TACTCTAGTT
TCGTTAATAT GGAGAGAGAT CTAGCTTCAG GACTCATTGG 2401 CCCTCTCCTC
ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461
CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA
2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC
CAGAGTTCCA 2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT
GATAGTTTGC AGTTGTCAGT 2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT
TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701 TTCTGTCTTC TTCTCTGGAT
ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761 CCTATTCCCA
TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821
TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC
2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA
TTTCAGCATA 2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC
TCCCAGAATT CAAGACACCC 3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC
CACAATTCCA GAAAATGACA TAGAGAAGAC 3061 TGACCCTTGG TTTGCACACA
GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121 TTTGTTGATG
CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181
AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA
3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG
ACATGGTATT 3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA
CTGGGGACAA CTGCAGCAAC 3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC
TAGTACATCA AATAATCTGA TTTCAACAAT 3421 TCCATCAGAC AATTTGGCAG
CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481 GCCAGTTCAT
TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541
TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA
3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT
CAACAGAGAG 3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT
TTGTTGACTA AAGATAATGC 3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA
GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781 TAATAGAAAG ACTCACATTG
ATGGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG 3841 GCAAAATATA
TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901
AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC
3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC
CACCAGATGC 4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG
CCAGAATCAG CAAGGTGGAT 4081 ACAAAGGACT CATGGAAAGA ACTCTCTGAA
CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141 AGTATCCTTA GGACCAGAAA
AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201 AGTGGTAGTA
GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261
AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA
4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC
AAGAGAATGT 4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT
TTCATGAAGA ACCTTTTCTT 4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC
ATATGACGGG GCATATGCTC CAGTACTTCA 4501 AGATTTTAGG TCATTAAATG
ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561 AAAAAAAGGG
GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621
GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA
4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG
AACTTGAAAA 4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA
AACATGAAAC ATTTGACCCC 4801 GAGCACCCTC ACACAGATAG ACTACAATGA
GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861 CTTATCAGAT TGCCTTACGA
GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921 CATTGCAAAG
GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981
CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA
5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG
CCATTCTAAC 5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG
GGGACAAGTG CCACAAATTC 5161 AGTCACATAC AAGAAAGTTG AGAACACTGT
TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221 TGGCAAAGTT GAATTGCTTC
CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281 AACTAGCAAT
GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341
AGAGGGAGCG ATTAAGTGGA ATGAAGCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT
5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG
CTTGGGATAA 5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC
CAAGAGAAGT CACCAGAAAA 5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT
GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581 AATAGCAGCA ATAAATGAGG
GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA 5641 AGGTAGGACT
GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701
AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG ATACCATATC
5761 AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC
AGAGCCCCCG 5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA
GTGGAGAGGC TCTGGGATTA 5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG
AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941 GTTCAAGAAA GTTGTTTTCC
AGGAATTTAC TGATGGCTCC TTTACTCAGC CCTTATACCG 6001 TGGAGAACTA
AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA 6061
TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT
6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT
TTGTCAAGCC 6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT
ATGGCACCCA CTAAAGATGA 6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC
TGATGTTGAC CTGGAAAAAG ATGTGCACTC 6301 AGGCCTGATT GGACCCCTTC
TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG 6361 ACAAGTGACA
GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG 6421
GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA
6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA
TGGATACACT 6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG
TATCTGCTCA GCATGGGCAG 6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG
TGGACATGTG TTCACTGTAC GAAAAAAAGA 6661 GGAGTATAAA ATGGCACTGT
ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT 6721 ACCATCCAAA
GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATGCTGG 6781
GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC
6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT
GGGCCCCAAA 6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG
AGCACCAAGG AGCCCTTTTC 6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT
GATTATTCAC GGCATCAAGA CCCAGGGTGC 7021 CCGTCAGAAG TTCTCCAGCC
TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG 7081 GAAGAAGTGG
CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA 7141
TGTGGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT
7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT
TGATGGGCTG 7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT
AAAGCAATAT CAGATGCACA 7321 GATTACTGCT TCATCCTACT TTACCAATAT
GTTTGCCACC TGGTCTCCTT CAAAAGCTCG 7381 ACTTCACCTC CAAGGGAGGA
GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG 7441 GCTGCAAGTG
GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA 7501
ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA
7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA
ATCAAGACTC 7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG
ACTCGCTACC TTCGAATTCA 7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT
GAGGATGGAG GTTCTGGGCT GCGAGGCACA 7741 GGACCTCTAC GACAAAACTC
ACACATGCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG 7801 ACCGTCAGTC
TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 7861
TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG
7921 GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG
AGCAGTACAA 7981 CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC
CAGGACTGGC TGAATGGCAA 8041 GGAGTACAAG TGCAAGGTCT CCAACAAAGC
CCTCCCAGCC CCCATCGAGA AAACCATCTC 8101 CAAAGCCAAA GGGCAGCCCC
GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 8161 GCTGACCAAG
AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 8221
CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT
8281 GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA
AGAGCAGGTG 8341 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG
GCTCTGCACA ACCACTACAC 8401 GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A (ii)
Fc (SEQ ID NO: 3)
TABLE-US-00007 TABLE 7 Polypeptide Sequence: FVIII-Fc A. B-Domain
Deleted FVIII-Fc Monomer Hybrid (BDD FVIIIFc monomer dimer):
created by coexpressing BDD FVIIIFc and Fc chains. Construct =
HC-LC-Fc fusion. An Fc expression cassette is cotransfected with
BDDFVIII-Fc to generate the BDD FVIIIFc monomer-. For the BDD
FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is
shown in double underline; remaining B domain sequence is shown in
italics. Signal peptides are underlined. i) B domain deleted
FVIII-Fc chain (19 amino acid signal sequence underlined) (SEQ ID
NO: 6) FVIII SIGNAL PEPTIDE: -19 MQIELSTCFFLCLLRFCFS FVIII MATURE
POLYPEPTIDE SEQUENCE: ##STR00001## ##STR00002## ##STR00003##
LKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYG
MSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQ
ASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKD
VHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKE
NYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGV
FETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKL
ARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGN
STGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAIS
DAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTS
MYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRME
VLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ii) Fc chain (20 amino acid
heterologous signal peptide from mouse Ig.kappa. chain underlined)
(SEQ ID NO: 4) FC SIGNAL PEPTIDE: -20 METDTLLLWVLLLWVPGSTG FC
SEQUENCE:
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK B. Full-length FVIIIFc monomer hybrid
(Full-length FVIIIFc monomer dimer): created by coexpressing
FVIIIFc and Fc chains. Construct = HC-B-LC-Fc fusion. An Fc
expression cassette is cotransfected with full-length FVIII-Fc to
generate the full-length FVIIIFc monomer. For the FVIIIFc chain,
the Fc sequence is shown in bold; HC sequence is shown in double
underline; B domain sequence is shown, in italics. Signal peptides
are underlined. i) Full-length FVIIIFc chain (FVIII signal peptide
underlined (SEQ ID NO: 8) FVIII SIGNAL PEPTIDE: -19
MQIELSTCFFLCLLRFCFS FVIII MATURE SEQUENCE: ##STR00004##
##STR00005## ##STR00006##
PSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETF
SDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNN
LISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGL
MNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSL
LIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPD
AQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGE
FTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFM
KNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYA
CTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEK
EKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGV
QESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLP
KVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDP
LAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRT
ERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI
AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVE
DNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWA
YFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCN
IQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYK
MALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITAS
GQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLD
GKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSM
PLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVT
TQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQS
WVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ii) Fc chain (20 amino
acid heterologous signal peptide from mouse Ig.kappa. chain
underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTG
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK
TABLE-US-00008 TABLE 8 Additional Sequences >CTP peptide 1 SEQ
ID NO: 9 DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL >CTP peptide 2 SEQ ID
NO: 10 SSSSKAPPPSLPSPSRLPGPSDTPILPQ >PAS peptide 1 SEQ ID NO: 11
ASPAAPAPASPAAPAPSAPA >PAS peptide 2 SEQ ID NO: 12
AAPASPAPAAPSAPAPAAPS >PAS peptide 3 SEQ ID NO: 13
APSSPSPSAPSSPSPASPSS >PAS peptide 4 SEQ ID NO: 14
APSSPSPSAPSSPSPASPS >PAS peptide 5 SEQ ID NO: 15
SSPSAPSPSSPASPSPSSPA >PAS peptide 6 SEQ ID NO: 16
AASPAAPSAPPAAASPAAPSAPPA >PAS peptide 7 SEQ ID NO: 17
ASAAAPAAASAAASAPSAAA >Albumin Binding Peptide Core Sequence SEQ
ID NO: 18 DICLPRWGCLW >GFP protein sequence (Genbank ID
AAG34521.1) SEQ ID NO: 19
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL
VTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV
NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD
HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSR
TSGSPGLQEFDIKLIDTVDLESCN >Example: Single-chain Human IgG1 Fc.
(Fc sequences with Gly/Ser linker underlined.) SEQ ID NO: 20
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVENAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGG
GSGGGGSDKTHTCPPCPAPELLGGPSVFLFPRKPKDTLMISRTPEVTCVVVDVSHEDPEV
KENWYVDGVEVHNAKTKPREEQYNSTYRVVgVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTOINKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALPINHYTQKSLSLSPGK >Mature
human albumin protein sequence (derived from NCBI Ref. Sequence
NP_000468) SEQ ID NO: 21
RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCV
ADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPR
LVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK
AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKL
VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVE
NDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYE
TTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKK
VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT
KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVK
HKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL >Albumin
binding peptide 1 SEQ ID NO: 22 RLIEDICLPRWGCLWEDD >Albumin
binding peptide 2 SEQ ID NO: 23 QRLMEDICLPRWGCLWEDDF >Albumin
binding peptide 3 SEQ ID NO: 24 QGLIGDICLPRWGCLWGDSVK >Albumin
binding peptide 4 SEQ ID NO: 25 GEWWEDICLPRWGCLWEEED
>Cysteine-containing peptide SEQ ID NO: 26 GGGSGCGGGS >Human
LRP1 sequence (signal peptide and transmembrane segment underlined;
NCBI Reference Sequence: CAA32112) SEQ ID NO: 27
MLTPPLLLLLPLLSALVAAAIDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSDEA
PEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQDCMDGSDEGRHCRELQGNCSRLGC
QHHCVPTLDGPTCYCNSSFQLQADOKTCKDFDECSVYGTCSQLCTNTDGSFICGCVEGYL
LQPDNRSCKAKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQTTAMDFSYANE
TVCWVHVGDSAAQTQLKCARMPGLKGFVDEHTINISLSLHHVEQMAIDWLTGNFYFVDDI
DDRIFVCNRNGDTCVTLLDLELYAPKGIALDPAMGKVEFTDYGQIPKVERCDMDGQNRTK
LVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVVDYEGKGRQTIIQGILIEHLYGLTVFE
NYLYATNSDNANAQQKTSVIRVNRFNSTEYQVVTRVDKGGALHIYHQRRQPRVRSHACEN
DQYGKPGGCSDICLLANSHKARTCRCRSGFSLGSDGKSCKKPEHELFLVYGKGRPGIIRG
MDMGAKVPDEHMIPIENLMNPRALDFHAETGFIYFADTTSYLIGROCIDGTERETILKDG
IHNVEGVAVDWMGDNLYWTDDGPKKTISVARLEKAAQTRKTLIEGKMTHPRAIVVDPLNG
WMYWTDWEEDPKDSRRGRLERAWMDGSHRDIFVTSKTVLWPNGLSLDIPAGRLYWVDAFY
DRIETILLNGTDRKIVYEGPELNHAFGLCHHONYLFWTEYRSGSVYRLERGVGGAPPTVT
LLRSERPPIFEIRMYDAQQQQVGTNKCRVNNGGCSSLCLATRGSRQCACAEDQVLDADGV
TCLANPSYVPPPQCQPGEFACANSRCIQERWKCDGDNDCLDNSDEAPALCHQHTCPSDRF
KCENNRCIPNRWLCDGDNDCGNSEDESNATCSARTCPPNQFSCASGRCIPISWTCDLDDD
CGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQF
KCNSGRCIPEHWTCDGDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRW
RCDGDTDCMDSSDEKSCEGVTHVCDRSVKFGOKDSARCISKAWVCDGDNDCEDNSDEENC
ESLACRPPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNNGGCSHNCSVAP
GEGIVCSCPLGMELGPDNHTCQIQSYCAKHLKCSQKCDQNKFSVKCSCYEGWVLEPDGES
CRSLDPFKPFIIFSNRHEIRRIDLHKGDYSVLVPGLRNTIALDFHLSQSALYWTDVVEDK
IYRGKLLDNGALTSFEVVIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGTLRTT
LLAGDIEHPRAIALDPRDGILFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWPNGLT
VDYLEKRILWIDARSDAIYSARYDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWTDWRTNT
LAKANKWTGHNVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPCSHLCLINYNRT
VSCACPHLMKLHKDNTTCYEFKKFLLYARQMEIRGVDLDAPYYNYIISFTVPDIDNVTVL
DYDAREQRVYWSDVRTQAIKRAFINGTGVETVVSADLPNAHGLAVDWVSRNLFWTSYDTN
KKQINVARLDGSFKNAVVQGLEQPHGLVVHPLRGKLYWTDGDNISMANMDGSNRTLLFSG
QKGPVGLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKATALAIMGDKLW
WADQVSEKMGTCSKADGSGSVVLRNSTTLVMHMKVYDESIQLDHKGTWPCSVNNGDCSQL
CLPTSETTRSCMCTAGYSLRSGQQACEGVGSFLLYSVHEGIRGIPLDPNDKSDALVPVSG
TSLAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWREDVVTNGIGRVEGIAVDWIAGNIY
WTDQGFDVIEVARLNGSFRYVVISQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSRLDG
TERVVLVNVSISWPNGISVDYQDGKLYWCDARTDKIERIDLETGENREVVLSSNNMDMES
VSVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVENRDRQKGTNVCA
VANGGCQQLCLYRGRGQRACACAHGMLAEDGASCREYAGYLLYSERTILKSIHLSDERNL
NAPVQPFEDPEHMKNVIALAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVE
NVGSVEGLAYHROWDTLYWTSYTTSTITRHTVDQTRPGAFERETVITMSGDDHPRAFVLD
ECQNLMFWTNWNEQHPSIMRAALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDK
IERCEYDGSHRYVILKSEPVHPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDI
PQQPMGIIAVANDTNSCELSPCRINNOGCQDLCLLTHQGHVNCSORGGRILQDDLTCRAV
NSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVS
NMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEMNCS
ATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPROPLNY
FACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLCDGSDDCG
DGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNSTCDD
REFMCQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWECDG
ENDCHDQSDEAPKNPECTSPEHKCNASSQFLCSSGRCVAEALLONGQDDCGDSSDERGCH
INECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADVDECSTTFPCSQRCINTH
GSYKCLCVEGYAPRGGDPHSCKAVTDEEPFLIFANRYYLRKLNLDGSNYTLLKQGLNNAV
ALDFDYREQMTYWTDVTTQGSMIRRMHLNGSNVQVLHRTGLSNPDGLAVDWVGGNLYWCD
KGRDTIEVSKLNGAYRTVLVSSGLREPRALVVDVQNGYLYWTDWGDHSLIGRIGMDGSSR
SVIVDTKITWPNGLTLDYVTERIYWADAREDYIEFASLDGSNRHVVLSQDIPHIFALTLF
EDYVYWTDWETKSINRAHKTTGTNKTLLISTLHRPMDLHVFHALRQPDVPNHPCKVNNGG
CSNLCLLSPGGGHKCACPTNFYLGSDGRTCVSNCTASQFVCKNDKCIPFWWKCDTEDDCG
DHSDEPPDCPEFKGRPGQFQCSTGICTNPAFICDGDNDCQDNSDEANCDIHVCLPSQFKC
TNTNRCIPGIFRCNGQDNCGDGEDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCV
DGSDEPANCTQMTCGVDEFRCKDSGRCIPARWKCDGEDDCGDGSDEPKEECDERTCEPYQ
FRCKNNROVPGRWQCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDC
ADGSDEKDCTPRCDMDQFQCKSGHCIPLRWRODADADCMDGSDEEACGTGVRTCPLDEFQ
CNNTLCKPLAWKCDGEDDCGDNSDENPEECARFVCPPNRPFRCKNDRVCLWIGRQCDGTD
NCGDGTDEEDCEPPIAHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEEDCSIDP
KLTSCATNASICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLREGTCSQLCNN
TKGGHLCSCARNFMKTHNTCKAEGSEYQVLYIADDNEIRELFPGHPHSAYEQAFQGDESV
RIDAMDVHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDROVTHLNISOLKMP
RGIAIDWVAGNVYWTDSGRDVIEVAQMKGENRKTLISGMIDEPHAIVVDPLRGTMYWSDW
GNHPKIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPI
VAADSKRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTGGLSHASDVVLYH
QHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPTPPPDAPRPGTC
NLQCFNGGSCFLNARRQPKCRCQPRYTGDKCELDQCWEHCRNGGTCAASPSGMPTCRCPT
GFTGPKCTQQVCAGYCANNSTCTVNQGNQPQCRCLPGFLGDRCQYRQCSGYCENFGTCQM
AADGSRQCRCTAYFEGSRCEVNKCSRCLEGACVVNKQSGDVTCNCTDGRVAPSCLTCVGH
CSNGGSCTMNSKMMPECQCPPHMTGPRCEEHVFSQQQPGHIASILIPLLLLLLLVLVAGV
VFWYKRRVQGAKGFQHQRMTNGAMNVE1GNPTYKMYEGGEPDDVGGLLDADFALDPDKPT
NFTNPVYATLYMGGHGSRHSLASTDEKRELLGRGPEDEIGDPLA >Biotin Acceptor
Peptide (BAP) SEQ ID NO: 28 LNDIFEAQKIEWH >Lipoate Acceptor
Peptide 2 (LAP2) SEQ ID NO: 29 GFEIDKVWYDLDA >HAPylation motif,
n = 1 to 400 SEQ ID NO: 30 (Gly4Ser)n >CTP SEQ ID NO: 31
DSSSSKAPPPSLPSPSRLPGPSDTPILPQ
Sequence CWU 1
1
3117583DNAArtificial SequencepSYN-FIX-030 1gcgcgcgttg acattgatta
ttgactagtt attaatagta atcaattacg gggtcattag 60ttcatagccc atatatggag
ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120gaccgcccaa
cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
180caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
gcccacttgg 240cagtacatca agtgtatcat atgccaagta cgccccctat
tgacgtcaat gacggtaaat 300ggcccgcctg gcattatgcc cagtacatga
ccttatggga ctttcctact tggcagtaca 360tctacgtatt agtcatcgct
attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420gtggatagcg
gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
480gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
tccgccccat 540tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
tataagcaga gctctctggc 600taactagaga acccactgct tactggctta
tcgaaattaa tacgactcac tatagggaga 660cccaagcttc gcgacgtacg
gccgccacca tgcagcgcgt gaacatgatc atggcagaat 720caccaggcct
catcaccatc tgccttttag gatatctact cagtgctgaa tgtacaggtt
780tgtttccttt tttaaaatac attgagtatg cttgcctttt agatatagaa
atatctgatg 840ctgtcttctt cactaaattt tgattacatg atttgacagc
aatattgaag agtctaacag 900ccagcacgca ggttggtaag tactgtggga
acatcacaga ttttggctcc atgccctaaa 960gagaaattgg ctttcagatt
atttggatta aaaacaaaga ctttcttaag agatgtaaaa 1020ttttcatgat
gttttctttt ttgctaaaac taaagaatta ttcttttaca tttcagtttt
1080tcttgatcat gaaaacgcca acaaaattct gaatcggcca aagaggtata
attcaggtaa 1140attggaagag tttgttcaag ggaatctaga gagagaatgt
atggaagaaa agtgtagttt 1200tgaagaagca cgagaagttt ttgaaaacac
tgaaagaaca actgaatttt ggaagcagta 1260tgttgatgga gatcagtgtg
agtccaatcc atgtttaaat ggcggcagtt gcaaggatga 1320cattaattcc
tatgaatgtt ggtgtccctt tggatttgaa ggaaagaact gtgaattaga
1380tgtaacatgt aacattaaga atggcagatg cgagcagttt tgtaaaaata
gtgctgataa 1440caaggtggtt tgctcctgta ctgagggata tcgacttgca
gaaaaccaga agtcctgtga 1500accagcagtg ccatttccat gtggaagagt
ttctgtttca caaacttcta agctcacccg 1560tgctgagact gtttttcctg
atgtggacta tgtaaattct actgaagctg aaaccatttt 1620ggataacatc
actcaaagca cccaatcatt taatgacttc actcgggttg ttggtggaga
1680agatgccaaa ccaggtcaat tcccttggca ggttgttttg aatggtaaag
ttgatgcatt 1740ctgtggaggc tctatcgtta atgaaaaatg gattgtaact
gctgcccact gtgttgaaac 1800tggtgttaaa attacagttg tcgcaggtga
acataatatt gaggagacag aacatacaga 1860gcaaaagcga aatgtgattc
gaattattcc tcaccacaac tacaatgcag ctattaataa 1920gtacaaccat
gacattgccc ttctggaact ggacgaaccc ttagtgctaa acagctacgt
1980tacacctatt tgcattgctg acaaggaata cacgaacatc ttcctcaaat
ttggatctgg 2040ctatgtaagt ggctggggaa gagtcttcca caaagggaga
tcagctttag ttcttcagta 2100ccttagagtt ccacttgttg accgagccac
atgtcttcga tctacaaagt tcaccatcta 2160taacaacatg ttctgtgctg
gcttccatga aggaggtaga gattcatgtc aaggagatag 2220tgggggaccc
catgttactg aagtggaagg gaccagtttc ttaactggaa ttattagctg
2280gggtgaagag tgtgcaatga aaggcaaata tggaatatat accaaggtgt
cccggtatgt 2340caactggatt aaggaaaaaa caaagctcac tgacaaaact
cacacatgcc caccgtgccc 2400agctccggaa ctcctgggcg gaccgtcagt
cttcctcttc cccccaaaac ccaaggacac 2460cctcatgatc tcccggaccc
ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga 2520ccctgaggtc
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa
2580gccgcgggag gagcagtaca acagcacgta ccgtgtggtc agcgtcctca
ccgtcctgca 2640ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc
tccaacaaag ccctcccagc 2700ccccatcgag aaaaccatct ccaaagccaa
agggcagccc cgagaaccac aggtgtacac 2760cctgccccca tcccgggatg
agctgaccaa gaaccaggtc agcctgacct gcctggtcaa 2820aggcttctat
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa
2880ctacaagacc acgcctcccg tgttggactc cgacggctcc ttcttcctct
acagcaagct 2940caccgtggac aagagcaggt ggcagcaggg gaacgtcttc
tcatgctccg tgatgcatga 3000ggctctgcac aaccactaca cgcagaagag
cctctccctg tctccgggta aatgagaatt 3060cagacatgat aagatacatt
gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 3120aatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
3180ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
gaggatcatc 3240cagccggcgt cccggaaaac gattccgaag cccaaccttt
catagaaggc ggcggtggaa 3300tcgaaatctc gtagcacgtg tcagtcctgc
tcctcggcca cgaagtgcac gcagttgccg 3360gccgggtcgc gcagggcgaa
ctcccgcccc cacggctgct cgccgatctc ggtcatggcc 3420ggcccggagg
cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg
3480tccaggccgc gcacccacac ccaggccagg gtgttgtccg gcaccacctg
gtcctggacc 3540gcgctgatga acagggtcac gtcgtcccgg accacaccgg
cgaagtcgtc ctccacgaag 3600tcccgggaga acccgagccg gtcggtccag
aactcgaccg ctccggcgac gtcgcgcgcg 3660gtgagcaccg gaacggcact
ggtcaacttg gccatggttt agttcctcac cttgtcgtat 3720tatactatgc
cgatatacta tgccgatgat taattgtcaa cacgtgctga tcagatccga
3780aaatggatat acaagctccc gggagctttt tgcaaaagcc taggcctcca
aaaaagcctc 3840ctcactactt ctggaatagc tcagaggcag aggcggcctc
ggcctctgca taaataaaaa 3900aaattagtca gccatggggc ggagaatggg
cggaactggg cggagttagg ggcgggatgg 3960gcggagttag gggcgggact
atggttgctg actaattgag atgcatgctt tgcatacttc 4020tgcctgctgg
ggagcctggg gactttccac acctggttgc tgactaattg agatgcatgc
4080tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctcgt
cgagctagct 4140tcgtgaggct ccggtgcccg tcagtgggca gagcgcacat
cgcccacagt ccccgagaag 4200ttggggggag gggtcggcaa ttgaaccggt
gcctagagaa ggtggcgcgg ggtaaactgg 4260gaaagtgatg tcgtgtactg
gctccgcctt tttcccgagg gtgggggaga accgtatata 4320agtgcagtag
tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
4380agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc
ttgcgtgcct 4440tgaattactt ccacctggct ccagtacgtg attcttgatc
ccgagctgga gccaggggcg 4500ggccttgcgc tttaggagcc ccttcgcctc
gtgcttgagt tgaggcctgg cctgggcgct 4560ggggccgccg cgtgcgaatc
tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 4620ctctagccat
ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
4680ttgtaaatgc gggccaggat ctgcacactg gtatttcggt ttttggggcc
gcgggcggcg 4740acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg
gggcctgcga gcgcggccac 4800cgagaatcgg acgggggtag tctcaagctg
gccggcctgc tctggtgcct ggcctcgcgc 4860cgccgtgtat cgccccgccc
tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 4920cggaaagatg
gccgcttccc ggccctgctc cagggggctc aaaatggagg acgcggcgct
4980cgggagagcg ggcgggtgag tcacccacac aaaggaaagg ggcctttccg
tcctcagccg 5040tcgcttcatg tgactccacg gagtaccggg cgccgtccag
gcacctcgat tagttctgga 5100gcttttggag tacgtcgtct ttaggttggg
gggaggggtt ttatgcgatg gagtttcccc 5160acactgagtg ggtggagact
gaagttaggc cagcttggca cttgatgtaa ttctccttgg 5220aatttgccct
ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
5280gtttttttct tccatttcag gtgtcgtgaa cacgtggtcg cggccgcgcc
gccaccatgg 5340agacagacac actcctgcta tgggtactgc tgctctgggt
tccaggttcc actggtgaca 5400aaactcacac atgcccaccg tgcccagcac
ctgaactcct gggaggaccg tcagtcttcc 5460tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag gtcacatgcg 5520tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac gtggacggcg
5580tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
acgtaccgtg 5640tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag tacaagtgca 5700aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa gccaaagggc 5760agccccgaga accacaggtg
tacaccctgc ccccatcccg cgatgagctg accaagaacc 5820aggtcagcct
gacctgcctg gtcaaaggct tctatcccag cgacatcgcc gtggagtggg
5880agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgttg
gactccgacg 5940gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag caggggaacg 6000tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag aagagcctct 6060ccctgtctcc gggtaaatga
ctcgagagat ctggccggct gggcccgttt cgaaggtaag 6120cctatcccta
accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac
6180cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
gccatctgtt 6240gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
ccactcccac tgtcctttcc 6300taataaaatg aggaaattgc atcgcattgt
ctgagtaggt gtcattctat tctggggggt 6360ggggtggggc aggacagcaa
gggggaggat tgggaagaca atagcaggca tgctggggat 6420gcggtgggct
ctatggcttc tgaggcggaa agaaccagtg gcggtaatac ggttatccac
6480agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
aggccaggaa 6540ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
cgcccccctg acgagcatca 6600caaaaatcga cgctcaagtc agaggtggcg
aaacccgaca ggactataaa gataccaggc 6660gtttccccct agaagctccc
tcgtgcgctc tcctgttccg accctgccgc ttaccggata 6720cctgtccgcc
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
6780tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
cccccgttca 6840gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
tccaacccgg taagacacga 6900cttatcgcca ctggcagcag ccactggtaa
caggattagc agagcgaggt atgtaggcgg 6960tgctacagag ttcttgaagt
ggtggcctaa ctacggctac actagaagaa cagtatttgg 7020tatctgcgct
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
7080caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
ttacgcgcag 7140aaaaaaagga tctcaagaag atcctttgat cttttctacg
gggtctgacg ctcagtggaa 7200cgaaaactca cgttaaggga ttttggtcat
gacattaacc tataaaaata ggcgtatcac 7260gaggcccttt cgtctcgcgc
gtttcggtga tgacggtgaa aacctctgac acatgcagct 7320cccggagacg
gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
7380cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
cagagcagat 7440tgtactgaga gtgcaccata tatgcggtgt gaaataccgc
acagatgcgt aaggagaaaa 7500taccgcatca ggcgccattc gccattcagg
ctgcgcaact gttgggaagg gcgatcggtg 7560cgggcctctt cgctattacg cca
75832688PRTArtificial SequenceFIX-Fc chain 2Met Gln Arg Val Asn Met
Ile Met Ala Glu Ser Pro Gly Leu Ile Thr 1 5 10 15 Ile Cys Leu Leu
Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30 Asp His
Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45
Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys 50
55 60 Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu
Asn 65 70 75 80 Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp
Gly Asp Gln 85 90 95 Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser
Cys Lys Asp Asp Ile 100 105 110 Asn Ser Tyr Glu Cys Trp Cys Pro Phe
Gly Phe Glu Gly Lys Asn Cys 115 120 125 Glu Leu Asp Val Thr Cys Asn
Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140 Cys Lys Asn Ser Ala
Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly 145 150 155 160 Tyr Arg
Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180
185 190 Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala
Glu 195 200 205 Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe
Asn Asp Phe 210 215 220 Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro
Gly Gln Phe Pro Trp 225 230 235 240 Gln Val Val Leu Asn Gly Lys Val
Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255 Val Asn Glu Lys Trp Ile
Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270 Val Lys Ile Thr
Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280 285 His Thr
Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295 300
Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu 305
310 315 320 Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile
Cys Ile 325 330 335 Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe
Gly Ser Gly Tyr 340 345 350 Val Ser Gly Trp Gly Arg Val Phe His Lys
Gly Arg Ser Ala Leu Val 355 360 365 Leu Gln Tyr Leu Arg Val Pro Leu
Val Asp Arg Ala Thr Cys Leu Arg 370 375 380 Ser Thr Lys Phe Thr Ile
Tyr Asn Asn Met Phe Cys Ala Gly Phe His 385 390 395 400 Glu Gly Gly
Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val 405 410 415 Thr
Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly 420 425
430 Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser
435 440 445 Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr Asp
Lys Thr 450 455 460 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser 465 470 475 480 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 485 490 495 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 500 505 510 Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 515 520 525 Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 530 535 540 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 545 550
555 560 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 565 570 575 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 580 585 590 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 595 600 605 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 610 615 620 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 625 630 635 640 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 645 650 655 Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 660 665 670
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 675
680 685 3 741DNAArtificial SequenceFc cassette from pSYN-FIX-030
3atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt
60gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggagg accgtcagtc
120ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc
tgaggtcaca 180tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca
agttcaactg gtacgtggac 240ggcgtggagg tgcataatgc caagacaaag
ccgcgggagg agcagtacaa cagcacgtac 300cgtgtggtca gcgtcctcac
cgtcctgcac caggactggc tgaatggcaa ggagtacaag 360tgcaaggtct
ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa
420gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgcgatga
gctgaccaag 480aaccaggtca gcctgacctg cctggtcaaa ggcttctatc
ccagcgacat cgccgtggag 540tgggagagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gttggactcc 600gacggctcct tcttcctcta
cagcaagctc accgtggaca agagcaggtg gcagcagggg 660aacgtcttct
catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc
720ctctccctgt ctccgggtaa a 7414247PRTArtificial SequenceFc chain
4Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1
5 10 15 Gly Ser Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro 20 25 30 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 50 55 60 Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95 Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125 Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135
140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
145 150 155 160 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser
Leu Ser Pro Gly Lys 245 55052DNAArtificial SequenceB-Domain Deleted
FVIIIFc 5atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg
ctttagtgcc 60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca
aagtgatctc 120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc
caaaatcttt tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt
gtagaattca cggatcacct tttcaacatc 240gctaagccaa ggccaccctg
gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg
tcattacact taagaacatg gcttcccatc ctgtcagtct
tcatgctgtt 360ggtgtatcct actggaaagc ttctgaggga gctgaatatg
atgatcagac cagtcaaagg 420gagaaagaag atgataaagt cttccctggt
ggaagccata catatgtctg gcaggtcctg 480aaagagaatg gtccaatggc
ctctgaccca ctgtgcctta cctactcata tctttctcat 540gtggacctgg
taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa
600gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact
ttttgctgta 660tttgatgaag ggaaaagttg gcactcagaa acaaagaact
ccttgatgca ggatagggat 720gctgcatctg ctcgggcctg gcctaaaatg
cacacagtca atggttatgt aaacaggtct 780ctgccaggtc tgattggatg
ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840accactcctg
aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat
900cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac
actcttgatg 960gaccttggac agtttctact gttttgtcat atctcttccc
accaacatga tggcatggaa 1020gcttatgtca aagtagacag ctgtccagag
gaaccccaac tacgaatgaa aaataatgaa 1080gaagcggaag actatgatga
tgatcttact gattctgaaa tggatgtggt caggtttgat 1140gatgacaact
ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact
1200tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt
agtcctcgcc 1260cccgatgaca gaagttataa aagtcaatat ttgaacaatg
gccctcagcg gattggtagg 1320aagtacaaaa aagtccgatt tatggcatac
acagatgaaa cctttaagac tcgtgaagct 1380attcagcatg aatcaggaat
cttgggacct ttactttatg gggaagttgg agacacactg 1440ttgattatat
ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact
1500gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt
gaaggatttt 1560ccaattctgc caggagaaat attcaaatat aaatggacag
tgactgtaga agatgggcca 1620actaaatcag atcctcggtg cctgacccgc
tattactcta gtttcgttaa tatggagaga 1680gatctagctt caggactcat
tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740agaggaaacc
agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag
1800aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc
agctggagtg 1860cagcttgagg atccagagtt ccaagcctcc aacatcatgc
acagcatcaa tggctatgtt 1920tttgatagtt tgcagttgtc agtttgtttg
catgaggtgg catactggta cattctaagc 1980attggagcac agactgactt
cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040atggtctatg
aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg
2100atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg
gaacagaggc 2160atgaccgcct tactgaaggt ttctagttgt gacaagaaca
ctggtgatta ttacgaggac 2220agttatgaag atatttcagc atacttgctg
agtaaaaaca atgccattga accaagaagc 2280ttctctcaaa acccaccagt
cttgaaacgc catcaacggg aaataactcg tactactctt 2340cagtcagatc
aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa
2400gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca
aaagaaaaca 2460cgacactatt ttattgctgc agtggagagg ctctgggatt
atgggatgag tagctcccca 2520catgttctaa gaaacagggc tcagagtggc
agtgtccctc agttcaagaa agttgttttc 2580caggaattta ctgatggctc
ctttactcag cccttatacc gtggagaact aaatgaacat 2640ttgggactcc
tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc
2700agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta
tgaggaagat 2760cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc
ctaatgaaac caaaacttac 2820ttttggaaag tgcaacatca tatggcaccc
actaaagatg agtttgactg caaagcctgg 2880gcttatttct ctgatgttga
cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940ctggtctgcc
acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa
3000tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac
tgaaaatatg 3060gaaagaaact gcagggctcc ctgcaatatc cagatggaag
atcccacttt taaagagaat 3120tatcgcttcc atgcaatcaa tggctacata
atggatacac tacctggctt agtaatggct 3180caggatcaaa ggattcgatg
gtatctgctc agcatgggca gcaatgaaaa catccattct 3240attcatttca
gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg
3300tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa
agctggaatt 3360tggcgggtgg aatgccttat tggcgagcat ctacatgctg
ggatgagcac actttttctg 3420gtgtacagca ataagtgtca gactcccctg
ggaatggctt ctggacacat tagagatttt 3480cagattacag cttcaggaca
atatggacag tgggccccaa agctggccag acttcattat 3540tccggatcaa
tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg
3600ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa
gttctccagc 3660ctctacatct ctcagtttat catcatgtat agtcttgatg
ggaagaagtg gcagacttat 3720cgaggaaatt ccactggaac cttaatggtc
ttctttggca atgtggattc atctgggata 3780aaacacaata tttttaaccc
tccaattatt gctcgataca tccgtttgca cccaactcat 3840tatagcattc
gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc
3900atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc
ttcatcctac 3960tttaccaata tgtttgccac ctggtctcct tcaaaagctc
gacttcacct ccaagggagg 4020agtaatgcct ggagacctca ggtgaataat
ccaaaagagt ggctgcaagt ggacttccag 4080aagacaatga aagtcacagg
agtaactact cagggagtaa aatctctgct taccagcatg 4140tatgtgaagg
agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt
4200cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc
tgtggtgaac 4260tctctagacc caccgttact gactcgctac cttcgaattc
acccccagag ttgggtgcac 4320cagattgccc tgaggatgga ggttctgggc
tgcgaggcac aggacctcta cgacaaaact 4380cacacatgcc caccgtgccc
agctccagaa ctcctgggcg gaccgtcagt cttcctcttc 4440cccccaaaac
ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg
4500gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga
cggcgtggag 4560gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
acagcacgta ccgtgtggtc 4620agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca aggagtacaa gtgcaaggtc 4680tccaacaaag ccctcccagc
ccccatcgag aaaaccatct ccaaagccaa agggcagccc 4740cgagaaccac
aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc
4800agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga
gtgggagagc 4860aatgggcagc cggagaacaa ctacaagacc acgcctcccg
tgttggactc cgacggctcc 4920ttcttcctct acagcaagct caccgtggac
aagagcaggt ggcagcaggg gaacgtcttc 4980tcatgctccg tgatgcatga
ggctctgcac aaccactaca cgcagaagag cctctccctg 5040tctccgggta aa
505261684PRTArtificial SequenceB domain deleted FVIII-Fc chain 6Met
Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10
15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp
Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn
Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr
Asp His Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro Pro Trp Met
Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val Tyr Asp Thr
Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 His Pro Val Ser
Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120 125 Glu Gly
Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp 130 135 140
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu 145
150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr
Tyr Ser 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn
Ser Gly Leu Ile 180 185 190 Gly Ala Leu Leu Val Cys Arg Glu Gly Ser
Leu Ala Lys Glu Lys Thr 195 200 205 Gln Thr Leu His Lys Phe Ile Leu
Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 Lys Ser Trp His Ser Glu
Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240 Ala Ala Ser
Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245 250 255 Val
Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val 260 265
270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln
Ala Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln
Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu Leu Phe Cys
His Ile Ser Ser His Gln His 325 330 335 Asp Gly Met Glu Ala Tyr Val
Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 Gln Leu Arg Met Lys
Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365 Leu Thr Asp
Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370 375 380 Pro
Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr 385 390
395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala
Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln
Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys
Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr Phe Lys Thr
Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu Gly Pro Leu
Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu Ile Ile Phe
Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490 495 His Gly
Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys 500 505 510
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe 515
520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser
Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn
Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu
Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg Gly Asn Gln
Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe Ser Val Phe
Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn Ile Gln Arg
Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615 620 Pro Glu
Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val 625 630 635
640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val
Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu
Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu Thr Val Phe
Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu Gly Cys His
Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr Ala Leu Leu
Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735 Tyr Tyr Glu
Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740 745 750 Asn
Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu 755 760
765 Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln
770 775 780 Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys
Lys Glu 785 790 795 800 Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln
Ser Pro Arg Ser Phe 805 810 815 Gln Lys Lys Thr Arg His Tyr Phe Ile
Ala Ala Val Glu Arg Leu Trp 820 825 830 Asp Tyr Gly Met Ser Ser Ser
Pro His Val Leu Arg Asn Arg Ala Gln 835 840 845 Ser Gly Ser Val Pro
Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 850 855 860 Asp Gly Ser
Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His 865 870 875 880
Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile 885
890 895 Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr
Ser 900 905 910 Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala
Glu Pro Arg 915 920 925 Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr
Tyr Phe Trp Lys Val 930 935 940 Gln His His Met Ala Pro Thr Lys Asp
Glu Phe Asp Cys Lys Ala Trp 945 950 955 960 Ala Tyr Phe Ser Asp Val
Asp Leu Glu Lys Asp Val His Ser Gly Leu 965 970 975 Ile Gly Pro Leu
Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His 980 985 990 Gly Arg
Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe 995 1000
1005 Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn
1010 1015 1020 Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr
Phe Lys 1025 1030 1035 Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr
Ile Met Asp Thr 1040 1045 1050 Leu Pro Gly Leu Val Met Ala Gln Asp
Gln Arg Ile Arg Trp Tyr 1055 1060 1065 Leu Leu Ser Met Gly Ser Asn
Glu Asn Ile His Ser Ile His Phe 1070 1075 1080 Ser Gly His Val Phe
Thr Val Arg Lys Lys Glu Glu Tyr Lys Met 1085 1090 1095 Ala Leu Tyr
Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met 1100 1105 1110 Leu
Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly 1115 1120
1125 Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser
1130 1135 1140 Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His
Ile Arg 1145 1150 1155 Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly
Gln Trp Ala Pro 1160 1165 1170 Lys Leu Ala Arg Leu His Tyr Ser Gly
Ser Ile Asn Ala Trp Ser 1175 1180 1185 Thr Lys Glu Pro Phe Ser Trp
Ile Lys Val Asp Leu Leu Ala Pro 1190 1195 1200 Met Ile Ile His Gly
Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe 1205 1210 1215 Ser Ser Leu
Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp 1220 1225 1230 Gly
Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu 1235 1240
1245 Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn
1250 1255 1260 Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu
His Pro 1265 1270 1275 Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met
Glu Leu Met Gly 1280 1285 1290 Cys Asp Leu Asn Ser Cys Ser Met Pro
Leu Gly Met Glu Ser Lys 1295 1300 1305 Ala Ile Ser Asp Ala Gln Ile
Thr Ala Ser Ser Tyr Phe Thr Asn 1310 1315 1320 Met Phe Ala Thr Trp
Ser Pro Ser Lys Ala Arg Leu His Leu Gln 1325 1330 1335 Gly Arg Ser
Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu 1340 1345 1350 Trp
Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val 1355 1360
1365 Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys
1370 1375 1380 Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp
Thr Leu 1385 1390 1395 Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln
Gly Asn Gln Asp 1400 1405 1410 Ser Phe Thr Pro Val Val Asn Ser Leu
Asp Pro Pro Leu Leu Thr 1415 1420 1425 Arg Tyr Leu Arg Ile His Pro
Gln Ser Trp Val His Gln Ile Ala 1430 1435 1440 Leu Arg Met Glu Val
Leu Gly Cys Glu Ala Gln Asp Leu Tyr Asp 1445 1450 1455 Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1460 1465 1470 Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 1475 1480
1485 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
1490 1495 1500 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly 1505 1510 1515 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr 1520 1525 1530 Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 1535 1540
1545 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
1550 1555 1560 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 1565 1570 1575 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp 1580 1585 1590 Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly 1595 1600 1605 Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln 1610 1615 1620 Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 1625 1630 1635 Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 1640 1645 1650 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 1655 1660
1665 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
1670 1675 1680 Lys 77734DNAArtificial SequenceFull-length FVIIIFc
7atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc
60accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc
120ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt
tccattcaac 180acctcagtcg tgtacaaaaa gactctgttt gtagaattca
cggatcacct tttcaacatc 240gctaagccaa ggccaccctg gatgggtctg
ctaggtccta ccatccaggc tgaggtttat 300gatacagtgg tcattacact
taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360ggtgtatcct
actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg
420gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg
gcaggtcctg 480aaagagaatg gtccaatggc ctctgaccca ctgtgcctta
cctactcata tctttctcat 540gtggacctgg taaaagactt gaattcaggc
ctcattggag ccctactagt atgtagagaa 600gggagtctgg ccaaggaaaa
gacacagacc ttgcacaaat ttatactact ttttgctgta 660tttgatgaag
ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat
720gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt
aaacaggtct 780ctgccaggtc tgattggatg ccacaggaaa tcagtctatt
ggcatgtgat tggaatgggc 840accactcctg aagtgcactc aatattcctc
gaaggtcaca catttcttgt gaggaaccat 900cgccaggcgt ccttggaaat
ctcgccaata actttcctta ctgctcaaac actcttgatg 960gaccttggac
agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa
1020gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa
aaataatgaa 1080gaagcggaag actatgatga tgatcttact gattctgaaa
tggatgtggt caggtttgat 1140gatgacaact ctccttcctt tatccaaatt
cgctcagttg ccaagaagca tcctaaaact 1200tgggtacatt acattgctgc
tgaagaggag gactgggact atgctccctt agtcctcgcc 1260cccgatgaca
gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg
1320aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac
tcgtgaagct 1380attcagcatg aatcaggaat cttgggacct ttactttatg
gggaagttgg agacacactg 1440ttgattatat ttaagaatca agcaagcaga
ccatataaca tctaccctca cggaatcact 1500gatgtccgtc ctttgtattc
aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560ccaattctgc
caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca
1620actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa
tatggagaga 1680gatctagctt caggactcat tggccctctc ctcatctgct
acaaagaatc tgtagatcaa 1740agaggaaacc agataatgtc agacaagagg
aatgtcatcc tgttttctgt atttgatgag 1800aaccgaagct ggtacctcac
agagaatata caacgctttc tccccaatcc agctggagtg 1860cagcttgagg
atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt
1920tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta
cattctaagc 1980attggagcac agactgactt cctttctgtc ttcttctctg
gatatacctt caaacacaaa 2040atggtctatg aagacacact caccctattc
ccattctcag gagaaactgt cttcatgtcg 2100atggaaaacc caggtctatg
gattctgggg tgccacaact cagactttcg gaacagaggc 2160atgaccgcct
tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac
2220agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga
accaagaagc 2280ttctcccaga attcaagaca ccctagcact aggcaaaagc
aatttaatgc caccacaatt 2340ccagaaaatg acatagagaa gactgaccct
tggtttgcac acagaacacc tatgcctaaa 2400atacaaaatg tctcctctag
tgatttgttg atgctcttgc gacagagtcc tactccacat 2460gggctatcct
tatctgatct ccaagaagcc aaatatgaga ctttttctga tgatccatca
2520cctggagcaa tagacagtaa taacagcctg tctgaaatga cacacttcag
gccacagctc 2580catcacagtg gggacatggt atttacccct gagtcaggcc
tccaattaag attaaatgag 2640aaactgggga caactgcagc aacagagttg
aagaaacttg atttcaaagt ttctagtaca 2700tcaaataatc tgatttcaac
aattccatca gacaatttgg cagcaggtac tgataataca 2760agttccttag
gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta
2820tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt
gagtgaagaa 2880aataatgatt caaagttgtt agaatcaggt ttaatgaata
gccaagaaag ttcatgggga 2940aaaaatgtat cgtcaacaga gagtggtagg
ttatttaaag ggaaaagagc tcatggacct 3000gctttgttga ctaaagataa
tgccttattc aaagttagca tctctttgtt aaagacaaac 3060aaaacttcca
ataattcagc aactaataga aagactcaca ttgatggccc atcattatta
3120attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga
gtttaaaaaa 3180gtgacacctt tgattcatga cagaatgctt atggacaaaa
atgctacagc tttgaggcta 3240aatcatatgt caaataaaac tacttcatca
aaaaacatgg aaatggtcca acagaaaaaa 3300gagggcccca ttccaccaga
tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360ttgccagaat
cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg
3420caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt
ggaaggtcag 3480aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg
gtgaatttac aaaggacgta 3540ggactcaaag agatggtttt tccaagcagc
agaaacctat ttcttactaa cttggataat 3600ttacatgaaa ataatacaca
caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660gaaacattaa
tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag
3720aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg
ttcatatgac 3780ggggcatatg ctccagtact tcaagatttt aggtcattaa
atgattcaac aaatagaaca 3840aagaaacaca cagctcattt ctcaaaaaaa
ggggaggaag aaaacttgga aggcttggga 3900aatcaaacca agcaaattgt
agagaaatat gcatgcacca caaggatatc tcctaataca 3960agccagcaga
attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca
4020ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac
ccagtggtcc 4080aaaaacatga aacatttgac cccgagcacc ctcacacaga
tagactacaa tgagaaggag 4140aaaggggcca ttactcagtc tcccttatca
gattgcctta cgaggagtca tagcatccct 4200caagcaaata gatctccatt
acccattgca aaggtatcat catttccatc tattagacct 4260atatatctga
ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat
4320agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc
caaaaaaaat 4380aacctttctt tagccattct aaccttggag atgactggtg
atcaaagaga ggttggctcc 4440ctggggacaa gtgccacaaa ttcagtcaca
tacaagaaag ttgagaacac tgttctcccg 4500aaaccagact tgcccaaaac
atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560cagaaggacc
tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg
4620gaagggagcc ttcttcaggg aacagaggga gcgattaagt ggaatgaagc
aaacagacct 4680ggaaaagttc cctttctgag agtagcaaca gaaagctctg
caaagactcc ctccaagcta 4740ttggatcctc ttgcttggga taaccactat
ggtactcaga taccaaaaga agagtggaaa 4800tcccaagaga agtcaccaga
aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860aacgcttgtg
aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa
4920atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca
aaacccacca 4980gtcttgaaac gccatcaacg ggaaataact cgtactactc
ttcagtcaga tcaagaggaa 5040attgactatg atgataccat atcagttgaa
atgaagaagg aagattttga catttatgat 5100gaggatgaaa atcagagccc
ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160gcagtggaga
ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg
5220gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt
tactgatggc 5280tcctttactc agcccttata ccgtggagaa ctaaatgaac
atttgggact cctggggcca 5340tatataagag cagaagttga agataatatc
atggtaactt tcagaaatca ggcctctcgt 5400ccctattcct tctattctag
ccttatttct tatgaggaag atcagaggca aggagcagaa 5460cctagaaaaa
actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat
5520catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt
ctctgatgtt 5580gacctggaaa aagatgtgca ctcaggcctg attggacccc
ttctggtctg ccacactaac 5640acactgaacc ctgctcatgg gagacaagtg
acagtacagg aatttgctct gtttttcacc 5700atctttgatg agaccaaaag
ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760ccctgcaata
tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc
5820aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca
aaggattcga 5880tggtatctgc tcagcatggg cagcaatgaa aacatccatt
ctattcattt cagtggacat 5940gtgttcactg tacgaaaaaa agaggagtat
aaaatggcac tgtacaatct ctatccaggt 6000gtttttgaga cagtggaaat
gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060attggcgagc
atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt
6120cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac
agcttcagga 6180caatatggac agtgggcccc aaagctggcc agacttcatt
attccggatc aatcaatgcc 6240tggagcacca aggagccctt ttcttggatc
aaggtggatc tgttggcacc aatgattatt 6300cacggcatca agacccaggg
tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360atcatcatgt
atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga
6420accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa
tatttttaac 6480cctccaatta ttgctcgata catccgtttg cacccaactc
attatagcat tcgcagcact 6540cttcgcatgg agttgatggg ctgtgattta
aatagttgca gcatgccatt gggaatggag 6600agtaaagcaa tatcagatgc
acagattact gcttcatcct actttaccaa tatgtttgcc 6660acctggtctc
cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct
6720caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat
gaaagtcaca 6780ggagtaacta ctcagggagt aaaatctctg cttaccagca
tgtatgtgaa ggagttcctc 6840atctccagca gtcaagatgg ccatcagtgg
actctctttt ttcagaatgg caaagtaaag 6900gtttttcagg gaaatcaaga
ctccttcaca cctgtggtga actctctaga cccaccgtta 6960ctgactcgct
accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg
7020gaggttctgg gctgcgaggc acaggacctc tacgacaaaa ctcacacatg
cccaccgtgc 7080ccagctccag aactcctggg cggaccgtca gtcttcctct
tccccccaaa acccaaggac 7140accctcatga tctcccggac ccctgaggtc
acatgcgtgg tggtggacgt gagccacgaa 7200gaccctgagg tcaagttcaa
ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 7260aagccgcggg
aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
7320caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca 7380gcccccatcg agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac 7440accctgcccc catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc 7500aaaggcttct atcccagcga
catcgccgtg gagtgggaga gcaatgggca gccggagaac 7560aactacaaga
ccacgcctcc cgtgttggac tccgacggct ccttcttcct ctacagcaag
7620ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc
cgtgatgcat 7680gaggctctgc acaaccacta cacgcagaag agcctctccc
tgtctccggg taaa 773482578PRTArtificial SequenceFull-length FVIIIFc
chain 8Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg
Phe 1 5 10 15 Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val
Glu Leu Ser 20 25 30 Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu
Pro Val Asp Ala Arg 35 40 45 Phe Pro Pro Arg Val Pro Lys Ser Phe
Pro Phe Asn Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val
Glu Phe Thr Asp His Leu Phe Asn Ile 65 70 75 80 Ala Lys Pro Arg Pro
Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln 85 90 95 Ala Glu Val
Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser 100 105 110 His
Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser 115 120
125 Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
130 135 140 Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln
Val Leu 145 150 155 160 Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu
Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser His Val Asp Leu Val Lys
Asp Leu Asn Ser Gly Leu Ile 180 185 190 Gly Ala Leu Leu Val Cys Arg
Glu Gly Ser Leu Ala Lys Glu Lys Thr 195 200 205 Gln Thr Leu His Lys
Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly 210 215 220 Lys Ser Trp
His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp 225 230 235 240
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr 245
250 255 Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser
Val 260 265 270 Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val
His Ser Ile 275 280 285 Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn
His Arg Gln Ala Ser 290 295 300 Leu Glu Ile Ser Pro Ile Thr Phe Leu
Thr Ala Gln Thr Leu Leu Met 305 310 315 320 Asp Leu Gly Gln Phe Leu
Leu Phe Cys His Ile Ser Ser His Gln His 325 330 335 Asp Gly Met Glu
Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro 340 345 350 Gln Leu
Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp 355 360 365
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser 370
375 380 Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys
Thr 385 390 395 400 Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp
Asp Tyr Ala Pro 405 410 415 Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr
Lys Ser Gln Tyr Leu Asn 420 425 430 Asn Gly Pro Gln Arg Ile Gly Arg
Lys Tyr Lys Lys Val Arg Phe Met 435 440 445 Ala Tyr Thr Asp Glu Thr
Phe Lys Thr Arg Glu Ala Ile Gln His Glu 450 455 460 Ser Gly Ile Leu
Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu 465 470 475 480 Leu
Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro 485 490
495 His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
500 505 510 Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu
Ile Phe 515 520 525 Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro
Thr Lys Ser Asp 530 535 540 Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser
Phe Val Asn Met Glu Arg 545 550 555 560 Asp Leu Ala Ser Gly Leu Ile
Gly Pro Leu Leu Ile Cys Tyr Lys Glu 565 570 575 Ser Val Asp Gln Arg
Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val 580 585 590 Ile Leu Phe
Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu 595 600 605 Asn
Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp 610 615
620 Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
625 630 635 640 Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val
Ala Tyr Trp 645 650 655 Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe
Leu Ser Val Phe Phe 660 665 670 Ser Gly Tyr Thr Phe Lys His Lys Met
Val Tyr Glu Asp Thr Leu Thr 675 680 685 Leu Phe Pro Phe Ser Gly Glu
Thr Val Phe Met Ser Met Glu Asn Pro 690 695 700 Gly Leu Trp Ile Leu
Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly 705 710 715 720 Met Thr
Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp 725 730 735
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys 740
745 750 Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His
Pro 755 760 765 Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro
Glu Asn Asp 770 775 780 Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg
Thr Pro Met Pro Lys 785 790 795 800 Ile Gln Asn Val Ser Ser Ser Asp
Leu Leu Met Leu Leu Arg Gln Ser 805 810 815 Pro Thr Pro His Gly Leu
Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr 820 825 830 Glu Thr Phe Ser
Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn 835 840 845 Ser Leu
Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly 850 855 860
Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu 865
870 875 880 Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp
Phe Lys 885 890 895 Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile
Pro Ser Asp Asn 900 905 910 Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser
Leu Gly Pro Pro Ser Met 915 920 925 Pro Val His Tyr Asp Ser Gln Leu
Asp Thr Thr Leu Phe Gly Lys Lys 930 935 940 Ser Ser Pro Leu Thr Glu
Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu 945 950 955
960 Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu
965 970 975 Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg
Leu Phe 980 985 990 Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr
Lys Asp Asn Ala 995 1000 1005 Leu Phe Lys Val Ser Ile Ser Leu Leu
Lys Thr Asn Lys Thr Ser 1010 1015 1020 Asn Asn Ser Ala Thr Asn Arg
Lys Thr His Ile Asp Gly Pro Ser 1025 1030 1035 Leu Leu Ile Glu Asn
Ser Pro Ser Val Trp Gln Asn Ile Leu Glu 1040 1045 1050 Ser Asp Thr
Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg 1055 1060 1065 Met
Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met 1070 1075
1080 Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln
1085 1090 1095 Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro
Asp Met 1100 1105 1110 Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser
Ala Arg Trp Ile 1115 1120 1125 Gln Arg Thr His Gly Lys Asn Ser Leu
Asn Ser Gly Gln Gly Pro 1130 1135 1140 Ser Pro Lys Gln Leu Val Ser
Leu Gly Pro Glu Lys Ser Val Glu 1145 1150 1155 Gly Gln Asn Phe Leu
Ser Glu Lys Asn Lys Val Val Val Gly Lys 1160 1165 1170 Gly Glu Phe
Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro 1175 1180 1185 Ser
Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu 1190 1195
1200 Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu
1205 1210 1215 Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro
Gln Ile 1220 1225 1230 His Thr Val Thr Gly Thr Lys Asn Phe Met Lys
Asn Leu Phe Leu 1235 1240 1245 Leu Ser Thr Arg Gln Asn Val Glu Gly
Ser Tyr Asp Gly Ala Tyr 1250 1255 1260 Ala Pro Val Leu Gln Asp Phe
Arg Ser Leu Asn Asp Ser Thr Asn 1265 1270 1275 Arg Thr Lys Lys His
Thr Ala His Phe Ser Lys Lys Gly Glu Glu 1280 1285 1290 Glu Asn Leu
Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu 1295 1300 1305 Lys
Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln 1310 1315
1320 Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg
1325 1330 1335 Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile
Val Asp 1340 1345 1350 Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys
His Leu Thr Pro 1355 1360 1365 Ser Thr Leu Thr Gln Ile Asp Tyr Asn
Glu Lys Glu Lys Gly Ala 1370 1375 1380 Ile Thr Gln Ser Pro Leu Ser
Asp Cys Leu Thr Arg Ser His Ser 1385 1390 1395 Ile Pro Gln Ala Asn
Arg Ser Pro Leu Pro Ile Ala Lys Val Ser 1400 1405 1410 Ser Phe Pro
Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe 1415 1420 1425 Gln
Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys 1430 1435
1440 Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys
1445 1450 1455 Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met
Thr Gly 1460 1465 1470 Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser
Ala Thr Asn Ser 1475 1480 1485 Val Thr Tyr Lys Lys Val Glu Asn Thr
Val Leu Pro Lys Pro Asp 1490 1495 1500 Leu Pro Lys Thr Ser Gly Lys
Val Glu Leu Leu Pro Lys Val His 1505 1510 1515 Ile Tyr Gln Lys Asp
Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser 1520 1525 1530 Pro Gly His
Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr 1535 1540 1545 Glu
Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val 1550 1555
1560 Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser
1565 1570 1575 Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly
Thr Gln 1580 1585 1590 Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys
Ser Pro Glu Lys 1595 1600 1605 Thr Ala Phe Lys Lys Lys Asp Thr Ile
Leu Ser Leu Asn Ala Cys 1610 1615 1620 Glu Ser Asn His Ala Ile Ala
Ala Ile Asn Glu Gly Gln Asn Lys 1625 1630 1635 Pro Glu Ile Glu Val
Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg 1640 1645 1650 Leu Cys Ser
Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu 1655 1660 1665 Ile
Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr 1670 1675
1680 Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile
1685 1690 1695 Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln
Lys Lys 1700 1705 1710 Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg
Leu Trp Asp Tyr 1715 1720 1725 Gly Met Ser Ser Ser Pro His Val Leu
Arg Asn Arg Ala Gln Ser 1730 1735 1740 Gly Ser Val Pro Gln Phe Lys
Lys Val Val Phe Gln Glu Phe Thr 1745 1750 1755 Asp Gly Ser Phe Thr
Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu 1760 1765 1770 His Leu Gly
Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp 1775 1780 1785 Asn
Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser 1790 1795
1800 Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly
1805 1810 1815 Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr
Lys Thr 1820 1825 1830 Tyr Phe Trp Lys Val Gln His His Met Ala Pro
Thr Lys Asp Glu 1835 1840 1845 Phe Asp Cys Lys Ala Trp Ala Tyr Phe
Ser Asp Val Asp Leu Glu 1850 1855 1860 Lys Asp Val His Ser Gly Leu
Ile Gly Pro Leu Leu Val Cys His 1865 1870 1875 Thr Asn Thr Leu Asn
Pro Ala His Gly Arg Gln Val Thr Val Gln 1880 1885 1890 Glu Phe Ala
Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp 1895 1900 1905 Tyr
Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn 1910 1915
1920 Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His
1925 1930 1935 Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu
Val Met 1940 1945 1950 Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu
Ser Met Gly Ser 1955 1960 1965 Asn Glu Asn Ile His Ser Ile His Phe
Ser Gly His Val Phe Thr 1970 1975 1980 Val Arg Lys Lys Glu Glu Tyr
Lys Met Ala Leu Tyr Asn Leu Tyr 1985 1990 1995 Pro Gly Val Phe Glu
Thr Val Glu Met Leu Pro Ser Lys Ala Gly 2000 2005 2010 Ile Trp Arg
Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly 2015 2020 2025 Met
Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro 2030 2035
2040 Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala
2045 2050 2055 Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg
Leu His 2060 2065 2070 Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys
Glu Pro Phe Ser 2075 2080 2085 Trp Ile Lys Val Asp Leu Leu Ala Pro
Met Ile Ile His Gly Ile 2090 2095 2100 Lys Thr Gln Gly Ala Arg Gln
Lys Phe Ser Ser Leu Tyr Ile Ser 2105 2110 2115 Gln Phe Ile Ile Met
Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr 2120 2125 2130 Tyr Arg Gly
Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn 2135 2140 2145 Val
Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile 2150 2155
2160 Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg
2165 2170 2175 Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn
Ser Cys 2180 2185 2190 Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile
Ser Asp Ala Gln 2195 2200 2205 Ile Thr Ala Ser Ser Tyr Phe Thr Asn
Met Phe Ala Thr Trp Ser 2210 2215 2220 Pro Ser Lys Ala Arg Leu His
Leu Gln Gly Arg Ser Asn Ala Trp 2225 2230 2235 Arg Pro Gln Val Asn
Asn Pro Lys Glu Trp Leu Gln Val Asp Phe 2240 2245 2250 Gln Lys Thr
Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys 2255 2260 2265 Ser
Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser 2270 2275
2280 Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys
2285 2290 2295 Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro
Val Val 2300 2305 2310 Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr
Leu Arg Ile His 2315 2320 2325 Pro Gln Ser Trp Val His Gln Ile Ala
Leu Arg Met Glu Val Leu 2330 2335 2340 Gly Cys Glu Ala Gln Asp Leu
Tyr Asp Lys Thr His Thr Cys Pro 2345 2350 2355 Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 2360 2365 2370 Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 2375 2380 2385 Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 2390 2395
2400 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
2405 2410 2415 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val 2420 2425 2430 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 2435 2440 2445 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile 2450 2455 2460 Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 2465 2470 2475 Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 2480 2485 2490 Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 2495 2500 2505 Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 2510 2515
2520 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
2525 2530 2535 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val 2540 2545 2550 Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 2555 2560 2565 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 2570 2575 932PRTHomo sapiensMISC_FEATURECTP peptide 1 9Asp Pro
Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 1 5 10 15
Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 20
25 30 1028PRTHomo sapiensMISC_FEATURECTP peptide 2 10Ser Ser Ser
Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 1 5 10 15 Leu
Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 20 25 1120PRTArtificial
SequencePAS peptide 1 11Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro
Ala Ala Pro Ala Pro 1 5 10 15 Ser Ala Pro Ala 20 1220PRTArtificial
SequencePAS peptide 2 12Ala Ala Pro Ala Ser Pro Ala Pro Ala Ala Pro
Ser Ala Pro Ala Pro 1 5 10 15 Ala Ala Pro Ser 20 1320PRTArtificial
SequencePAS peptide 3 13Ala Pro Ser Ser Pro Ser Pro Ser Ala Pro Ser
Ser Pro Ser Pro Ala 1 5 10 15 Ser Pro Ser Ser 20 1419PRTArtificial
SequencePAS peptide 4 14Ala Pro Ser Ser Pro Ser Pro Ser Ala Pro Ser
Ser Pro Ser Pro Ala 1 5 10 15 Ser Pro Ser 1520PRTArtificial
SequencePAS peptide 5 15Ser Ser Pro Ser Ala Pro Ser Pro Ser Ser Pro
Ala Ser Pro Ser Pro 1 5 10 15 Ser Ser Pro Ala 20 1624PRTArtificial
SequencePAS peptide 6 16Ala Ala Ser Pro Ala Ala Pro Ser Ala Pro Pro
Ala Ala Ala Ser Pro 1 5 10 15 Ala Ala Pro Ser Ala Pro Pro Ala 20
1720PRTArtificial SequencePAS peptide 7 17Ala Ser Ala Ala Ala Pro
Ala Ala Ala Ser Ala Ala Ala Ser Ala Pro 1 5 10 15 Ser Ala Ala Ala
20 1811PRTArtificial SequenceAlbumin Binding Peptide Core Sequence
18Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10
19264PRTArtificial SequenceGFP protein 19Met Ser Lys Gly Glu Glu
Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60 Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys
Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180
185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu
Leu Tyr Lys Ser Arg 225 230 235 240 Thr Ser Gly Ser Pro Gly Leu Gln
Glu Phe Asp Ile Lys Leu Ile Asp 245 250 255 Thr Val Asp Leu Glu Ser
Cys Asn 260 20474PRTArtificial SequenceSingle-chain Human IgG1 Fc
20Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1
5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50
55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180
185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 210 215 220 Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Ser Asp
Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305
310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu 370 375 380 Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425
430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470
21591PRTHomo sapiensMISC_FEATUREMature human albumin protein
sequence 21Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala
His Arg 1 5 10 15 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu
Val Leu Ile Ala 20 25 30 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe
Glu Asp His Val Lys Leu 35 40 45 Val Asn Glu Val Thr Glu Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser 50 55 60 Ala Glu Asn Cys Asp Lys
Ser Leu His Thr Leu Phe Gly Asp Lys Leu 65 70 75 80 Cys Thr Val Ala
Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 85 90 95 Cys Ala
Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 100 105 110
Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 115
120 125 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys
Tyr 130 135 140 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala
Pro Glu Leu 145 150 155 160 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala
Phe Thr Glu Cys Cys Gln 165 170 175 Ala Ala Asp Lys Ala Ala Cys Leu
Leu Pro Lys Leu Asp Glu Leu Arg 180 185 190 Asp Glu Gly Lys Ala Ser
Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 195 200 205 Leu Gln Lys Phe
Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 210 215 220 Leu Ser
Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 225 230 235
240 Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu
245 250 255 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile
Cys Glu 260 265 270 Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys
Cys Glu Lys Pro 275 280 285 Leu Leu Glu Lys Ser His Cys Ile Ala Glu
Val Glu Asn Asp Glu Met 290 295 300 Pro Ala Asp Leu Pro Ser Leu Ala
Ala Asp Phe Val Glu Ser Lys Asp 305 310 315 320 Val Cys Lys Asn Tyr
Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 325 330 335 Leu Tyr Glu
Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 340 345 350 Leu
Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 355 360
365 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
370 375 380 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu 385 390 395 400 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg 405 410 415 Tyr Thr Lys Lys Val Pro Gln Val Ser
Thr Pro Thr Leu Val Glu Val 420 425 430 Ser Arg Asn Leu Gly Lys Val
Gly Ser Lys Cys Cys Lys His Pro Glu 435 440 445 Ala Lys Arg Met Pro
Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 450 455 460 Gln Leu Cys
Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 465 470 475 480
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 485
490 495 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr 500 505 510 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln 515 520 525 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val
Lys His Lys Pro Lys 530 535 540 Ala Thr Lys Glu Gln Leu Lys Ala Val
Met Asp Asp Phe Ala Ala Phe 545 550 555 560 Val Glu Lys Cys Cys Lys
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 565 570 575 Glu Gly Lys Lys
Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 590
2218PRTArtificial SequenceAlbumin binding peptide 1 22Arg Leu Ile
Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp
Asp 2320PRTArtificial SequenceAlbumin binding peptide 2 23Gln Arg
Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15
Glu Asp Asp Phe 20 2421PRTArtificial SequenceAlbumin binding
peptide 3 24Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys
Leu Trp 1 5 10 15 Gly Asp Ser Val Lys 20 2520PRTArtificial
SequenceAlbumin binding peptide 4 25Gly Glu Trp Trp Glu Asp Ile Cys
Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 15 Glu Glu Glu Asp 20
2610PRTArtificial SequenceCysteine-containing peptide 26Gly Gly Gly
Ser Gly Cys Gly Gly Gly Ser 1 5 10 274544PRTHomo
sapiensMISC_FEATUREHuman LRP1 27Met Leu Thr Pro Pro Leu Leu Leu Leu
Leu Pro Leu Leu Ser Ala Leu 1 5 10 15 Val Ala Ala Ala Ile Asp Ala
Pro Lys Thr Cys Ser Pro Lys Gln Phe 20 25 30 Ala Cys Arg Asp Gln
Ile Thr Cys Ile Ser Lys Gly Trp Arg Cys Asp 35 40 45 Gly Glu Arg
Asp Cys Pro Asp Gly Ser Asp Glu Ala Pro Glu Ile Cys 50 55 60 Pro
Gln Ser Lys Ala Gln Arg Cys Gln Pro Asn Glu His Asn Cys Leu 65 70
75 80 Gly Thr Glu Leu Cys Val Pro Met Ser Arg Leu Cys Asn Gly Val
Gln 85 90 95 Asp Cys Met Asp Gly Ser Asp Glu Gly Pro His Cys Arg
Glu Leu Gln 100 105 110 Gly Asn Cys Ser Arg Leu Gly Cys Gln His His
Cys Val Pro Thr Leu 115 120 125 Asp Gly Pro Thr Cys Tyr Cys Asn Ser
Ser Phe Gln Leu Gln Ala Asp 130 135 140 Gly Lys Thr Cys Lys Asp Phe
Asp Glu Cys Ser Val Tyr Gly Thr Cys 145 150 155 160 Ser Gln Leu Cys
Thr Asn Thr Asp Gly Ser Phe Ile Cys Gly Cys Val 165 170 175 Glu Gly
Tyr Leu Leu Gln Pro Asp Asn Arg Ser Cys Lys Ala Lys Asn 180 185 190
Glu Pro Val Asp Arg Pro Pro Val Leu Leu Ile Ala Asn Ser Gln Asn 195
200 205 Ile Leu Ala Thr Tyr Leu Ser Gly Ala Gln Val Ser Thr Ile Thr
Pro 210 215 220 Thr Ser Thr Arg Gln Thr Thr Ala Met Asp Phe Ser Tyr
Ala Asn Glu 225 230 235 240 Thr Val Cys Trp Val His Val Gly Asp Ser
Ala Ala Gln Thr Gln Leu 245 250 255 Lys Cys Ala Arg Met Pro Gly Leu
Lys Gly Phe Val Asp Glu His Thr 260 265 270 Ile Asn Ile Ser Leu Ser
Leu His His Val Glu Gln Met Ala Ile Asp 275 280 285 Trp Leu Thr Gly
Asn Phe Tyr Phe Val Asp Asp Ile Asp Asp Arg Ile 290 295 300 Phe Val
Cys Asn Arg Asn Gly Asp Thr Cys Val Thr Leu Leu Asp Leu 305 310 315
320 Glu Leu Tyr Asn Pro Lys Gly Ile Ala Leu Asp Pro Ala Met Gly Lys
325 330 335 Val Phe Phe Thr Asp Tyr Gly Gln Ile Pro Lys Val Glu Arg
Cys Asp 340 345 350 Met Asp Gly Gln Asn Arg Thr Lys Leu Val Asp Ser
Lys Ile Val Phe 355 360 365 Pro His Gly Ile Thr Leu Asp Leu Val Ser
Arg Leu Val Tyr Trp Ala 370 375 380 Asp Ala Tyr Leu Asp Tyr Ile Glu
Val Val Asp Tyr Glu Gly Lys Gly 385 390 395 400 Arg Gln Thr Ile Ile
Gln Gly Ile Leu Ile Glu His Leu Tyr Gly Leu 405 410 415 Thr Val Phe
Glu Asn Tyr Leu Tyr Ala Thr Asn Ser Asp Asn Ala Asn 420 425 430 Ala
Gln Gln Lys Thr Ser Val Ile Arg Val Asn Arg Phe Asn Ser Thr 435 440
445 Glu Tyr Gln Val Val Thr Arg Val Asp Lys Gly Gly Ala Leu His Ile
450 455 460 Tyr His Gln Arg Arg Gln Pro Arg Val Arg Ser His Ala Cys
Glu Asn 465 470 475 480 Asp Gln Tyr Gly Lys Pro Gly Gly Cys Ser Asp
Ile Cys Leu Leu Ala 485 490 495 Asn Ser His Lys Ala Arg Thr Cys Arg
Cys Arg Ser Gly Phe Ser Leu 500 505 510 Gly Ser Asp Gly Lys Ser Cys
Lys Lys Pro Glu His Glu Leu Phe Leu 515 520 525 Val Tyr Gly Lys Gly
Arg Pro Gly Ile Ile Arg Gly Met Asp Met Gly 530 535 540 Ala Lys Val
Pro Asp Glu His Met Ile Pro Ile Glu Asn Leu Met Asn 545 550 555 560
Pro Arg Ala Leu Asp Phe His Ala Glu Thr Gly Phe Ile Tyr Phe Ala 565
570 575 Asp Thr Thr Ser Tyr Leu Ile Gly Arg Gln Lys Ile Asp Gly Thr
Glu 580 585 590 Arg Glu Thr Ile Leu Lys Asp Gly Ile His Asn Val Glu
Gly Val Ala 595 600 605 Val Asp Trp Met Gly Asp Asn Leu Tyr Trp Thr
Asp Asp Gly Pro Lys 610 615 620 Lys Thr Ile Ser Val Ala Arg Leu Glu
Lys Ala Ala Gln Thr Arg Lys 625 630 635 640 Thr Leu Ile Glu Gly Lys
Met Thr His Pro Arg Ala Ile Val Val Asp 645 650 655 Pro Leu Asn Gly
Trp Met Tyr Trp Thr Asp Trp Glu Glu Asp Pro Lys 660 665 670 Asp Ser
Arg Arg Gly Arg Leu Glu Arg Ala Trp Met Asp Gly Ser His 675 680 685
Arg Asp Ile Phe Val Thr Ser Lys Thr Val Leu Trp Pro Asn Gly Leu 690
695 700 Ser Leu Asp Ile Pro Ala Gly Arg Leu Tyr Trp Val Asp Ala Phe
Tyr 705 710 715 720 Asp Arg Ile Glu Thr Ile Leu Leu Asn Gly Thr Asp
Arg Lys Ile Val 725 730 735 Tyr Glu Gly Pro Glu Leu Asn His Ala Phe
Gly Leu Cys His His Gly 740 745 750 Asn Tyr Leu Phe Trp Thr Glu Tyr
Arg Ser Gly Ser Val Tyr Arg Leu 755 760 765 Glu Arg Gly Val Gly Gly
Ala Pro Pro Thr Val Thr Leu Leu Arg Ser 770 775 780 Glu Arg Pro Pro
Ile Phe Glu Ile Arg Met Tyr Asp Ala Gln Gln Gln 785 790 795 800 Gln
Val Gly Thr Asn Lys Cys Arg Val Asn Asn Gly Gly Cys Ser Ser 805 810
815 Leu Cys Leu Ala Thr Pro Gly Ser Arg Gln Cys Ala Cys Ala Glu Asp
820 825 830 Gln Val Leu Asp Ala Asp Gly Val Thr Cys Leu Ala Asn Pro
Ser Tyr 835 840 845 Val Pro Pro Pro Gln Cys Gln Pro Gly Glu Phe Ala
Cys Ala Asn Ser 850 855 860 Arg Cys Ile Gln Glu Arg Trp Lys Cys Asp
Gly Asp Asn Asp Cys Leu 865 870 875 880 Asp Asn Ser Asp Glu Ala Pro
Ala Leu Cys His Gln His Thr Cys Pro 885 890 895 Ser Asp Arg Phe Lys
Cys Glu Asn Asn Arg Cys Ile Pro Asn Arg Trp 900 905 910 Leu Cys Asp
Gly Asp Asn Asp Cys Gly Asn Ser Glu Asp Glu Ser Asn 915 920 925 Ala
Thr Cys Ser Ala Arg Thr Cys Pro Pro Asn Gln Phe Ser Cys Ala 930 935
940 Ser Gly Arg Cys Ile Pro Ile Ser Trp Thr Cys Asp Leu Asp Asp Asp
945 950 955 960 Cys Gly Asp Arg Ser Asp Glu Ser Ala Ser Cys Ala Tyr
Pro Thr Cys 965 970 975 Phe Pro Leu Thr Gln Phe Thr Cys Asn Asn Gly
Arg Cys Ile Asn Ile 980 985 990 Asn Trp Arg Cys Asp Asn Asp Asn Asp
Cys Gly Asp Asn Ser Asp Glu 995 1000 1005 Ala Gly Cys Ser His Ser
Cys Ser Ser Thr Gln Phe Lys Cys Asn 1010 1015 1020 Ser Gly Arg Cys
Ile Pro Glu His Trp Thr Cys Asp Gly Asp Asn 1025 1030 1035 Asp Cys
Gly Asp Tyr Ser Asp Glu Thr His Ala Asn Cys Thr Asn 1040 1045 1050
Gln Ala Thr Arg Pro Pro Gly Gly Cys His Thr Asp Glu Phe Gln 1055
1060 1065 Cys Arg Leu Asp Gly Leu Cys Ile Pro Leu Arg Trp Arg Cys
Asp 1070 1075 1080 Gly Asp Thr Asp Cys Met Asp Ser Ser Asp Glu Lys
Ser Cys Glu 1085 1090 1095 Gly Val Thr His Val Cys Asp Pro Ser Val
Lys Phe Gly Cys Lys 1100 1105 1110 Asp Ser Ala Arg Cys Ile Ser Lys
Ala Trp Val Cys Asp Gly Asp 1115 1120 1125 Asn Asp Cys Glu Asp Asn
Ser Asp Glu Glu Asn Cys Glu Ser Leu 1130 1135 1140 Ala Cys Arg Pro
Pro Ser His
Pro Cys Ala Asn Asn Thr Ser Val 1145 1150 1155 Cys Leu Pro Pro Asp
Lys Leu Cys Asp Gly Asn Asp Asp Cys Gly 1160 1165 1170 Asp Gly Ser
Asp Glu Gly Glu Leu Cys Asp Gln Cys Ser Leu Asn 1175 1180 1185 Asn
Gly Gly Cys Ser His Asn Cys Ser Val Ala Pro Gly Glu Gly 1190 1195
1200 Ile Val Cys Ser Cys Pro Leu Gly Met Glu Leu Gly Pro Asp Asn
1205 1210 1215 His Thr Cys Gln Ile Gln Ser Tyr Cys Ala Lys His Leu
Lys Cys 1220 1225 1230 Ser Gln Lys Cys Asp Gln Asn Lys Phe Ser Val
Lys Cys Ser Cys 1235 1240 1245 Tyr Glu Gly Trp Val Leu Glu Pro Asp
Gly Glu Ser Cys Arg Ser 1250 1255 1260 Leu Asp Pro Phe Lys Pro Phe
Ile Ile Phe Ser Asn Arg His Glu 1265 1270 1275 Ile Arg Arg Ile Asp
Leu His Lys Gly Asp Tyr Ser Val Leu Val 1280 1285 1290 Pro Gly Leu
Arg Asn Thr Ile Ala Leu Asp Phe His Leu Ser Gln 1295 1300 1305 Ser
Ala Leu Tyr Trp Thr Asp Val Val Glu Asp Lys Ile Tyr Arg 1310 1315
1320 Gly Lys Leu Leu Asp Asn Gly Ala Leu Thr Ser Phe Glu Val Val
1325 1330 1335 Ile Gln Tyr Gly Leu Ala Thr Pro Glu Gly Leu Ala Val
Asp Trp 1340 1345 1350 Ile Ala Gly Asn Ile Tyr Trp Val Glu Ser Asn
Leu Asp Gln Ile 1355 1360 1365 Glu Val Ala Lys Leu Asp Gly Thr Leu
Arg Thr Thr Leu Leu Ala 1370 1375 1380 Gly Asp Ile Glu His Pro Arg
Ala Ile Ala Leu Asp Pro Arg Asp 1385 1390 1395 Gly Ile Leu Phe Trp
Thr Asp Trp Asp Ala Ser Leu Pro Arg Ile 1400 1405 1410 Glu Ala Ala
Ser Met Ser Gly Ala Gly Arg Arg Thr Val His Arg 1415 1420 1425 Glu
Thr Gly Ser Gly Gly Trp Pro Asn Gly Leu Thr Val Asp Tyr 1430 1435
1440 Leu Glu Lys Arg Ile Leu Trp Ile Asp Ala Arg Ser Asp Ala Ile
1445 1450 1455 Tyr Ser Ala Arg Tyr Asp Gly Ser Gly His Met Glu Val
Leu Arg 1460 1465 1470 Gly His Glu Phe Leu Ser His Pro Phe Ala Val
Thr Leu Tyr Gly 1475 1480 1485 Gly Glu Val Tyr Trp Thr Asp Trp Arg
Thr Asn Thr Leu Ala Lys 1490 1495 1500 Ala Asn Lys Trp Thr Gly His
Asn Val Thr Val Val Gln Arg Thr 1505 1510 1515 Asn Thr Gln Pro Phe
Asp Leu Gln Val Tyr His Pro Ser Arg Gln 1520 1525 1530 Pro Met Ala
Pro Asn Pro Cys Glu Ala Asn Gly Gly Gln Gly Pro 1535 1540 1545 Cys
Ser His Leu Cys Leu Ile Asn Tyr Asn Arg Thr Val Ser Cys 1550 1555
1560 Ala Cys Pro His Leu Met Lys Leu His Lys Asp Asn Thr Thr Cys
1565 1570 1575 Tyr Glu Phe Lys Lys Phe Leu Leu Tyr Ala Arg Gln Met
Glu Ile 1580 1585 1590 Arg Gly Val Asp Leu Asp Ala Pro Tyr Tyr Asn
Tyr Ile Ile Ser 1595 1600 1605 Phe Thr Val Pro Asp Ile Asp Asn Val
Thr Val Leu Asp Tyr Asp 1610 1615 1620 Ala Arg Glu Gln Arg Val Tyr
Trp Ser Asp Val Arg Thr Gln Ala 1625 1630 1635 Ile Lys Arg Ala Phe
Ile Asn Gly Thr Gly Val Glu Thr Val Val 1640 1645 1650 Ser Ala Asp
Leu Pro Asn Ala His Gly Leu Ala Val Asp Trp Val 1655 1660 1665 Ser
Arg Asn Leu Phe Trp Thr Ser Tyr Asp Thr Asn Lys Lys Gln 1670 1675
1680 Ile Asn Val Ala Arg Leu Asp Gly Ser Phe Lys Asn Ala Val Val
1685 1690 1695 Gln Gly Leu Glu Gln Pro His Gly Leu Val Val His Pro
Leu Arg 1700 1705 1710 Gly Lys Leu Tyr Trp Thr Asp Gly Asp Asn Ile
Ser Met Ala Asn 1715 1720 1725 Met Asp Gly Ser Asn Arg Thr Leu Leu
Phe Ser Gly Gln Lys Gly 1730 1735 1740 Pro Val Gly Leu Ala Ile Asp
Phe Pro Glu Ser Lys Leu Tyr Trp 1745 1750 1755 Ile Ser Ser Gly Asn
His Thr Ile Asn Arg Cys Asn Leu Asp Gly 1760 1765 1770 Ser Gly Leu
Glu Val Ile Asp Ala Met Arg Ser Gln Leu Gly Lys 1775 1780 1785 Ala
Thr Ala Leu Ala Ile Met Gly Asp Lys Leu Trp Trp Ala Asp 1790 1795
1800 Gln Val Ser Glu Lys Met Gly Thr Cys Ser Lys Ala Asp Gly Ser
1805 1810 1815 Gly Ser Val Val Leu Arg Asn Ser Thr Thr Leu Val Met
His Met 1820 1825 1830 Lys Val Tyr Asp Glu Ser Ile Gln Leu Asp His
Lys Gly Thr Asn 1835 1840 1845 Pro Cys Ser Val Asn Asn Gly Asp Cys
Ser Gln Leu Cys Leu Pro 1850 1855 1860 Thr Ser Glu Thr Thr Arg Ser
Cys Met Cys Thr Ala Gly Tyr Ser 1865 1870 1875 Leu Arg Ser Gly Gln
Gln Ala Cys Glu Gly Val Gly Ser Phe Leu 1880 1885 1890 Leu Tyr Ser
Val His Glu Gly Ile Arg Gly Ile Pro Leu Asp Pro 1895 1900 1905 Asn
Asp Lys Ser Asp Ala Leu Val Pro Val Ser Gly Thr Ser Leu 1910 1915
1920 Ala Val Gly Ile Asp Phe His Ala Glu Asn Asp Thr Ile Tyr Trp
1925 1930 1935 Val Asp Met Gly Leu Ser Thr Ile Ser Arg Ala Lys Arg
Asp Gln 1940 1945 1950 Thr Trp Arg Glu Asp Val Val Thr Asn Gly Ile
Gly Arg Val Glu 1955 1960 1965 Gly Ile Ala Val Asp Trp Ile Ala Gly
Asn Ile Tyr Trp Thr Asp 1970 1975 1980 Gln Gly Phe Asp Val Ile Glu
Val Ala Arg Leu Asn Gly Ser Phe 1985 1990 1995 Arg Tyr Val Val Ile
Ser Gln Gly Leu Asp Lys Pro Arg Ala Ile 2000 2005 2010 Thr Val His
Pro Glu Lys Gly Tyr Leu Phe Trp Thr Glu Trp Gly 2015 2020 2025 Gln
Tyr Pro Arg Ile Glu Arg Ser Arg Leu Asp Gly Thr Glu Arg 2030 2035
2040 Val Val Leu Val Asn Val Ser Ile Ser Trp Pro Asn Gly Ile Ser
2045 2050 2055 Val Asp Tyr Gln Asp Gly Lys Leu Tyr Trp Cys Asp Ala
Arg Thr 2060 2065 2070 Asp Lys Ile Glu Arg Ile Asp Leu Glu Thr Gly
Glu Asn Arg Glu 2075 2080 2085 Val Val Leu Ser Ser Asn Asn Met Asp
Met Phe Ser Val Ser Val 2090 2095 2100 Phe Glu Asp Phe Ile Tyr Trp
Ser Asp Arg Thr His Ala Asn Gly 2105 2110 2115 Ser Ile Lys Arg Gly
Ser Lys Asp Asn Ala Thr Asp Ser Val Pro 2120 2125 2130 Leu Arg Thr
Gly Ile Gly Val Gln Leu Lys Asp Ile Lys Val Phe 2135 2140 2145 Asn
Arg Asp Arg Gln Lys Gly Thr Asn Val Cys Ala Val Ala Asn 2150 2155
2160 Gly Gly Cys Gln Gln Leu Cys Leu Tyr Arg Gly Arg Gly Gln Arg
2165 2170 2175 Ala Cys Ala Cys Ala His Gly Met Leu Ala Glu Asp Gly
Ala Ser 2180 2185 2190 Cys Arg Glu Tyr Ala Gly Tyr Leu Leu Tyr Ser
Glu Arg Thr Ile 2195 2200 2205 Leu Lys Ser Ile His Leu Ser Asp Glu
Arg Asn Leu Asn Ala Pro 2210 2215 2220 Val Gln Pro Phe Glu Asp Pro
Glu His Met Lys Asn Val Ile Ala 2225 2230 2235 Leu Ala Phe Asp Tyr
Arg Ala Gly Thr Ser Pro Gly Thr Pro Asn 2240 2245 2250 Arg Ile Phe
Phe Ser Asp Ile His Phe Gly Asn Ile Gln Gln Ile 2255 2260 2265 Asn
Asp Asp Gly Ser Arg Arg Ile Thr Ile Val Glu Asn Val Gly 2270 2275
2280 Ser Val Glu Gly Leu Ala Tyr His Arg Gly Trp Asp Thr Leu Tyr
2285 2290 2295 Trp Thr Ser Tyr Thr Thr Ser Thr Ile Thr Arg His Thr
Val Asp 2300 2305 2310 Gln Thr Arg Pro Gly Ala Phe Glu Arg Glu Thr
Val Ile Thr Met 2315 2320 2325 Ser Gly Asp Asp His Pro Arg Ala Phe
Val Leu Asp Glu Cys Gln 2330 2335 2340 Asn Leu Met Phe Trp Thr Asn
Trp Asn Glu Gln His Pro Ser Ile 2345 2350 2355 Met Arg Ala Ala Leu
Ser Gly Ala Asn Val Leu Thr Leu Ile Glu 2360 2365 2370 Lys Asp Ile
Arg Thr Pro Asn Gly Leu Ala Ile Asp His Arg Ala 2375 2380 2385 Glu
Lys Leu Tyr Phe Ser Asp Ala Thr Leu Asp Lys Ile Glu Arg 2390 2395
2400 Cys Glu Tyr Asp Gly Ser His Arg Tyr Val Ile Leu Lys Ser Glu
2405 2410 2415 Pro Val His Pro Phe Gly Leu Ala Val Tyr Gly Glu His
Ile Phe 2420 2425 2430 Trp Thr Asp Trp Val Arg Arg Ala Val Gln Arg
Ala Asn Lys His 2435 2440 2445 Val Gly Ser Asn Met Lys Leu Leu Arg
Val Asp Ile Pro Gln Gln 2450 2455 2460 Pro Met Gly Ile Ile Ala Val
Ala Asn Asp Thr Asn Ser Cys Glu 2465 2470 2475 Leu Ser Pro Cys Arg
Ile Asn Asn Gly Gly Cys Gln Asp Leu Cys 2480 2485 2490 Leu Leu Thr
His Gln Gly His Val Asn Cys Ser Cys Arg Gly Gly 2495 2500 2505 Arg
Ile Leu Gln Asp Asp Leu Thr Cys Arg Ala Val Asn Ser Ser 2510 2515
2520 Cys Arg Ala Gln Asp Glu Phe Glu Cys Ala Asn Gly Glu Cys Ile
2525 2530 2535 Asn Phe Ser Leu Thr Cys Asp Gly Val Pro His Cys Lys
Asp Lys 2540 2545 2550 Ser Asp Glu Lys Pro Ser Tyr Cys Asn Ser Arg
Arg Cys Lys Lys 2555 2560 2565 Thr Phe Arg Gln Cys Ser Asn Gly Arg
Cys Val Ser Asn Met Leu 2570 2575 2580 Trp Cys Asn Gly Ala Asp Asp
Cys Gly Asp Gly Ser Asp Glu Ile 2585 2590 2595 Pro Cys Asn Lys Thr
Ala Cys Gly Val Gly Glu Phe Arg Cys Arg 2600 2605 2610 Asp Gly Thr
Cys Ile Gly Asn Ser Ser Arg Cys Asn Gln Phe Val 2615 2620 2625 Asp
Cys Glu Asp Ala Ser Asp Glu Met Asn Cys Ser Ala Thr Asp 2630 2635
2640 Cys Ser Ser Tyr Phe Arg Leu Gly Val Lys Gly Val Leu Phe Gln
2645 2650 2655 Pro Cys Glu Arg Thr Ser Leu Cys Tyr Ala Pro Ser Trp
Val Cys 2660 2665 2670 Asp Gly Ala Asn Asp Cys Gly Asp Tyr Ser Asp
Glu Arg Asp Cys 2675 2680 2685 Pro Gly Val Lys Arg Pro Arg Cys Pro
Leu Asn Tyr Phe Ala Cys 2690 2695 2700 Pro Ser Gly Arg Cys Ile Pro
Met Ser Trp Thr Cys Asp Lys Glu 2705 2710 2715 Asp Asp Cys Glu His
Gly Glu Asp Glu Thr His Cys Asn Lys Phe 2720 2725 2730 Cys Ser Glu
Ala Gln Phe Glu Cys Gln Asn His Arg Cys Ile Ser 2735 2740 2745 Lys
Gln Trp Leu Cys Asp Gly Ser Asp Asp Cys Gly Asp Gly Ser 2750 2755
2760 Asp Glu Ala Ala His Cys Glu Gly Lys Thr Cys Gly Pro Ser Ser
2765 2770 2775 Phe Ser Cys Pro Gly Thr His Val Cys Val Pro Glu Arg
Trp Leu 2780 2785 2790 Cys Asp Gly Asp Lys Asp Cys Ala Asp Gly Ala
Asp Glu Ser Ile 2795 2800 2805 Ala Ala Gly Cys Leu Tyr Asn Ser Thr
Cys Asp Asp Arg Glu Phe 2810 2815 2820 Met Cys Gln Asn Arg Gln Cys
Ile Pro Lys His Phe Val Cys Asp 2825 2830 2835 His Asp Arg Asp Cys
Ala Asp Gly Ser Asp Glu Ser Pro Glu Cys 2840 2845 2850 Glu Tyr Pro
Thr Cys Gly Pro Ser Glu Phe Arg Cys Ala Asn Gly 2855 2860 2865 Arg
Cys Leu Ser Ser Arg Gln Trp Glu Cys Asp Gly Glu Asn Asp 2870 2875
2880 Cys His Asp Gln Ser Asp Glu Ala Pro Lys Asn Pro His Cys Thr
2885 2890 2895 Ser Pro Glu His Lys Cys Asn Ala Ser Ser Gln Phe Leu
Cys Ser 2900 2905 2910 Ser Gly Arg Cys Val Ala Glu Ala Leu Leu Cys
Asn Gly Gln Asp 2915 2920 2925 Asp Cys Gly Asp Ser Ser Asp Glu Arg
Gly Cys His Ile Asn Glu 2930 2935 2940 Cys Leu Ser Arg Lys Leu Ser
Gly Cys Ser Gln Asp Cys Glu Asp 2945 2950 2955 Leu Lys Ile Gly Phe
Lys Cys Arg Cys Arg Pro Gly Phe Arg Leu 2960 2965 2970 Lys Asp Asp
Gly Arg Thr Cys Ala Asp Val Asp Glu Cys Ser Thr 2975 2980 2985 Thr
Phe Pro Cys Ser Gln Arg Cys Ile Asn Thr His Gly Ser Tyr 2990 2995
3000 Lys Cys Leu Cys Val Glu Gly Tyr Ala Pro Arg Gly Gly Asp Pro
3005 3010 3015 His Ser Cys Lys Ala Val Thr Asp Glu Glu Pro Phe Leu
Ile Phe 3020 3025 3030 Ala Asn Arg Tyr Tyr Leu Arg Lys Leu Asn Leu
Asp Gly Ser Asn 3035 3040 3045 Tyr Thr Leu Leu Lys Gln Gly Leu Asn
Asn Ala Val Ala Leu Asp 3050 3055 3060 Phe Asp Tyr Arg Glu Gln Met
Ile Tyr Trp Thr Asp Val Thr Thr 3065 3070 3075 Gln Gly Ser Met Ile
Arg Arg Met His Leu Asn Gly Ser Asn Val 3080 3085 3090 Gln Val Leu
His Arg Thr Gly Leu Ser Asn Pro Asp Gly Leu Ala 3095 3100 3105 Val
Asp Trp Val Gly Gly Asn Leu Tyr Trp Cys Asp Lys Gly Arg 3110 3115
3120 Asp Thr Ile Glu Val Ser Lys Leu Asn Gly Ala Tyr Arg Thr Val
3125 3130 3135 Leu Val Ser Ser Gly Leu Arg Glu Pro Arg Ala Leu Val
Val Asp 3140 3145 3150 Val Gln Asn Gly Tyr Leu Tyr Trp Thr Asp Trp
Gly Asp His Ser 3155 3160 3165 Leu Ile Gly Arg Ile Gly Met Asp Gly
Ser Ser Arg Ser Val Ile 3170 3175 3180 Val Asp Thr Lys Ile Thr Trp
Pro Asn Gly Leu Thr Leu Asp Tyr 3185 3190 3195 Val Thr Glu Arg Ile
Tyr Trp Ala Asp Ala Arg Glu Asp Tyr Ile 3200 3205 3210 Glu Phe Ala
Ser Leu Asp Gly Ser Asn Arg His Val Val Leu Ser 3215 3220 3225 Gln
Asp Ile Pro His Ile Phe Ala Leu Thr Leu Phe Glu Asp Tyr 3230 3235
3240 Val Tyr Trp Thr Asp Trp Glu Thr Lys Ser Ile Asn Arg Ala His
3245 3250 3255 Lys Thr Thr Gly Thr Asn Lys Thr Leu Leu Ile Ser Thr
Leu His 3260 3265 3270 Arg Pro Met Asp Leu His Val Phe His Ala Leu
Arg Gln Pro Asp 3275 3280 3285 Val Pro Asn His Pro Cys Lys Val Asn
Asn Gly Gly Cys Ser Asn 3290 3295 3300 Leu Cys Leu Leu Ser Pro Gly
Gly Gly His Lys Cys Ala Cys Pro 3305 3310 3315 Thr Asn Phe Tyr Leu
Gly Ser Asp Gly Arg Thr Cys Val Ser Asn 3320 3325 3330 Cys Thr Ala
Ser Gln Phe Val Cys Lys Asn Asp Lys Cys Ile Pro 3335
3340 3345 Phe Trp Trp Lys Cys Asp Thr Glu Asp Asp Cys Gly Asp His
Ser 3350 3355 3360 Asp Glu Pro Pro Asp Cys Pro Glu Phe Lys Cys Arg
Pro Gly Gln 3365 3370 3375 Phe Gln Cys Ser Thr Gly Ile Cys Thr Asn
Pro Ala Phe Ile Cys 3380 3385 3390 Asp Gly Asp Asn Asp Cys Gln Asp
Asn Ser Asp Glu Ala Asn Cys 3395 3400 3405 Asp Ile His Val Cys Leu
Pro Ser Gln Phe Lys Cys Thr Asn Thr 3410 3415 3420 Asn Arg Cys Ile
Pro Gly Ile Phe Arg Cys Asn Gly Gln Asp Asn 3425 3430 3435 Cys Gly
Asp Gly Glu Asp Glu Arg Asp Cys Pro Glu Val Thr Cys 3440 3445 3450
Ala Pro Asn Gln Phe Gln Cys Ser Ile Thr Lys Arg Cys Ile Pro 3455
3460 3465 Arg Val Trp Val Cys Asp Arg Asp Asn Asp Cys Val Asp Gly
Ser 3470 3475 3480 Asp Glu Pro Ala Asn Cys Thr Gln Met Thr Cys Gly
Val Asp Glu 3485 3490 3495 Phe Arg Cys Lys Asp Ser Gly Arg Cys Ile
Pro Ala Arg Trp Lys 3500 3505 3510 Cys Asp Gly Glu Asp Asp Cys Gly
Asp Gly Ser Asp Glu Pro Lys 3515 3520 3525 Glu Glu Cys Asp Glu Arg
Thr Cys Glu Pro Tyr Gln Phe Arg Cys 3530 3535 3540 Lys Asn Asn Arg
Cys Val Pro Gly Arg Trp Gln Cys Asp Tyr Asp 3545 3550 3555 Asn Asp
Cys Gly Asp Asn Ser Asp Glu Glu Ser Cys Thr Pro Arg 3560 3565 3570
Pro Cys Ser Glu Ser Glu Phe Ser Cys Ala Asn Gly Arg Cys Ile 3575
3580 3585 Ala Gly Arg Trp Lys Cys Asp Gly Asp His Asp Cys Ala Asp
Gly 3590 3595 3600 Ser Asp Glu Lys Asp Cys Thr Pro Arg Cys Asp Met
Asp Gln Phe 3605 3610 3615 Gln Cys Lys Ser Gly His Cys Ile Pro Leu
Arg Trp Arg Cys Asp 3620 3625 3630 Ala Asp Ala Asp Cys Met Asp Gly
Ser Asp Glu Glu Ala Cys Gly 3635 3640 3645 Thr Gly Val Arg Thr Cys
Pro Leu Asp Glu Phe Gln Cys Asn Asn 3650 3655 3660 Thr Leu Cys Lys
Pro Leu Ala Trp Lys Cys Asp Gly Glu Asp Asp 3665 3670 3675 Cys Gly
Asp Asn Ser Asp Glu Asn Pro Glu Glu Cys Ala Arg Phe 3680 3685 3690
Val Cys Pro Pro Asn Arg Pro Phe Arg Cys Lys Asn Asp Arg Val 3695
3700 3705 Cys Leu Trp Ile Gly Arg Gln Cys Asp Gly Thr Asp Asn Cys
Gly 3710 3715 3720 Asp Gly Thr Asp Glu Glu Asp Cys Glu Pro Pro Thr
Ala His Thr 3725 3730 3735 Thr His Cys Lys Asp Lys Lys Glu Phe Leu
Cys Arg Asn Gln Arg 3740 3745 3750 Cys Leu Ser Ser Ser Leu Arg Cys
Asn Met Phe Asp Asp Cys Gly 3755 3760 3765 Asp Gly Ser Asp Glu Glu
Asp Cys Ser Ile Asp Pro Lys Leu Thr 3770 3775 3780 Ser Cys Ala Thr
Asn Ala Ser Ile Cys Gly Asp Glu Ala Arg Cys 3785 3790 3795 Val Arg
Thr Glu Lys Ala Ala Tyr Cys Ala Cys Arg Ser Gly Phe 3800 3805 3810
His Thr Val Pro Gly Gln Pro Gly Cys Gln Asp Ile Asn Glu Cys 3815
3820 3825 Leu Arg Phe Gly Thr Cys Ser Gln Leu Cys Asn Asn Thr Lys
Gly 3830 3835 3840 Gly His Leu Cys Ser Cys Ala Arg Asn Phe Met Lys
Thr His Asn 3845 3850 3855 Thr Cys Lys Ala Glu Gly Ser Glu Tyr Gln
Val Leu Tyr Ile Ala 3860 3865 3870 Asp Asp Asn Glu Ile Arg Ser Leu
Phe Pro Gly His Pro His Ser 3875 3880 3885 Ala Tyr Glu Gln Ala Phe
Gln Gly Asp Glu Ser Val Arg Ile Asp 3890 3895 3900 Ala Met Asp Val
His Val Lys Ala Gly Arg Val Tyr Trp Thr Asn 3905 3910 3915 Trp His
Thr Gly Thr Ile Ser Tyr Arg Ser Leu Pro Pro Ala Ala 3920 3925 3930
Pro Pro Thr Thr Ser Asn Arg His Arg Arg Gln Ile Asp Arg Gly 3935
3940 3945 Val Thr His Leu Asn Ile Ser Gly Leu Lys Met Pro Arg Gly
Ile 3950 3955 3960 Ala Ile Asp Trp Val Ala Gly Asn Val Tyr Trp Thr
Asp Ser Gly 3965 3970 3975 Arg Asp Val Ile Glu Val Ala Gln Met Lys
Gly Glu Asn Arg Lys 3980 3985 3990 Thr Leu Ile Ser Gly Met Ile Asp
Glu Pro His Ala Ile Val Val 3995 4000 4005 Asp Pro Leu Arg Gly Thr
Met Tyr Trp Ser Asp Trp Gly Asn His 4010 4015 4020 Pro Lys Ile Glu
Thr Ala Ala Met Asp Gly Thr Leu Arg Glu Thr 4025 4030 4035 Leu Val
Gln Asp Asn Ile Gln Trp Pro Thr Gly Leu Ala Val Asp 4040 4045 4050
Tyr His Asn Glu Arg Leu Tyr Trp Ala Asp Ala Lys Leu Ser Val 4055
4060 4065 Ile Gly Ser Ile Arg Leu Asn Gly Thr Asp Pro Ile Val Ala
Ala 4070 4075 4080 Asp Ser Lys Arg Gly Leu Ser His Pro Phe Ser Ile
Asp Val Phe 4085 4090 4095 Glu Asp Tyr Ile Tyr Gly Val Thr Tyr Ile
Asn Asn Arg Val Phe 4100 4105 4110 Lys Ile His Lys Phe Gly His Ser
Pro Leu Val Asn Leu Thr Gly 4115 4120 4125 Gly Leu Ser His Ala Ser
Asp Val Val Leu Tyr His Gln His Lys 4130 4135 4140 Gln Pro Glu Val
Thr Asn Pro Cys Asp Arg Lys Lys Cys Glu Trp 4145 4150 4155 Leu Cys
Leu Leu Ser Pro Ser Gly Pro Val Cys Thr Cys Pro Asn 4160 4165 4170
Gly Lys Arg Leu Asp Asn Gly Thr Cys Val Pro Val Pro Ser Pro 4175
4180 4185 Thr Pro Pro Pro Asp Ala Pro Arg Pro Gly Thr Cys Asn Leu
Gln 4190 4195 4200 Cys Phe Asn Gly Gly Ser Cys Phe Leu Asn Ala Arg
Arg Gln Pro 4205 4210 4215 Lys Cys Arg Cys Gln Pro Arg Tyr Thr Gly
Asp Lys Cys Glu Leu 4220 4225 4230 Asp Gln Cys Trp Glu His Cys Arg
Asn Gly Gly Thr Cys Ala Ala 4235 4240 4245 Ser Pro Ser Gly Met Pro
Thr Cys Arg Cys Pro Thr Gly Phe Thr 4250 4255 4260 Gly Pro Lys Cys
Thr Gln Gln Val Cys Ala Gly Tyr Cys Ala Asn 4265 4270 4275 Asn Ser
Thr Cys Thr Val Asn Gln Gly Asn Gln Pro Gln Cys Arg 4280 4285 4290
Cys Leu Pro Gly Phe Leu Gly Asp Arg Cys Gln Tyr Arg Gln Cys 4295
4300 4305 Ser Gly Tyr Cys Glu Asn Phe Gly Thr Cys Gln Met Ala Ala
Asp 4310 4315 4320 Gly Ser Arg Gln Cys Arg Cys Thr Ala Tyr Phe Glu
Gly Ser Arg 4325 4330 4335 Cys Glu Val Asn Lys Cys Ser Arg Cys Leu
Glu Gly Ala Cys Val 4340 4345 4350 Val Asn Lys Gln Ser Gly Asp Val
Thr Cys Asn Cys Thr Asp Gly 4355 4360 4365 Arg Val Ala Pro Ser Cys
Leu Thr Cys Val Gly His Cys Ser Asn 4370 4375 4380 Gly Gly Ser Cys
Thr Met Asn Ser Lys Met Met Pro Glu Cys Gln 4385 4390 4395 Cys Pro
Pro His Met Thr Gly Pro Arg Cys Glu Glu His Val Phe 4400 4405 4410
Ser Gln Gln Gln Pro Gly His Ile Ala Ser Ile Leu Ile Pro Leu 4415
4420 4425 Leu Leu Leu Leu Leu Leu Val Leu Val Ala Gly Val Val Phe
Trp 4430 4435 4440 Tyr Lys Arg Arg Val Gln Gly Ala Lys Gly Phe Gln
His Gln Arg 4445 4450 4455 Met Thr Asn Gly Ala Met Asn Val Glu Ile
Gly Asn Pro Thr Tyr 4460 4465 4470 Lys Met Tyr Glu Gly Gly Glu Pro
Asp Asp Val Gly Gly Leu Leu 4475 4480 4485 Asp Ala Asp Phe Ala Leu
Asp Pro Asp Lys Pro Thr Asn Phe Thr 4490 4495 4500 Asn Pro Val Tyr
Ala Thr Leu Tyr Met Gly Gly His Gly Ser Arg 4505 4510 4515 His Ser
Leu Ala Ser Thr Asp Glu Lys Arg Glu Leu Leu Gly Arg 4520 4525 4530
Gly Pro Glu Asp Glu Ile Gly Asp Pro Leu Ala 4535 4540
2813PRTArtificial SequenceBiotin Acceptor Peptide (BAP) 28Leu Asn
Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His 1 5 10
2913PRTArtificial SequenceLipoate Acceptor Peptide 2 (LAP2) 29Gly
Phe Glu Ile Asp Lys Val Trp Tyr Asp Leu Asp Ala 1 5 10
305PRTArtificial SequenceHAPylation motif 30Gly Gly Gly Gly Ser 1 5
3129PRTArtificial SequenceCTP 31Asp Ser Ser Ser Ser Lys Ala Pro Pro
Pro Ser Leu Pro Ser Pro Ser 1 5 10 15 Arg Leu Pro Gly Pro Ser Asp
Thr Pro Ile Leu Pro Gln 20 25
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